JP2020523011A - Single-cell whole-genome library for methylation sequencing - Google Patents

Abstract

Provided herein are methods of preparing a sequencing library for determining the methylation status of nucleic acids from multiple single cells. The method combines split-and-pool combinatorial indexing with bisulfite treatment techniques to rapidly, accurately, and inexpensively characterize a large number of single-cell methylation profiles.

Description

CROSS REFERENCE TO RELATED APPLICATIONS This application claims the benefit of US Provisional Application No. 62/516,324, filed June 7, 2017, which is hereby incorporated by reference.

Embodiments of the disclosure relate to nucleic acid sequencing. In particular, embodiments of the methods and compositions provided herein relate to generating a single cell bisulfite sequencing library and obtaining sequence data therefrom.

High cell number single cell sequencing has demonstrated its effectiveness in segregating populations within complex tissues via transcriptome, chromatin accessibility, and mutation differences. Furthermore, single cell division allowed us to assess cell differentiation trajectories in a genome-specific pattern such as DNA methylation. DNA methylation is a covalent addition to cytosine and is a cell-type specific target for activity modification in tissue expression. DNA methylation can be probed by base pair resolution using deamination chemistry of sodium bisulfite treatment.

Recent studies have optimized bisulfite sequencing as long as single cell input is required, either in single cell reduced expression bisulfite sequencing (scRRBS) or single cell whole genome bisulfite sequencing (scWGBS). Turned into However, these methods lack scalability, relying on single cell deconvolution through parallel and separate library generation, where single cell reactions are performed separately. A whole new set of reagents is required for each cell sequencing, resulting in a linear scaling of the cost for each additional cell. Due to the challenge of bisulfite conversion of DNA, droplet- or chip-based microfluidic systems have not been developed for single-cell bisulfite sequencing, and theoretically using alternative platforms. There is no viable strategy.

Provided herein are compositions and expandable high cell number single cell methylome profiling assays. Single Cell Whole Genome Sequencing (scWGBS) is improved by the single cell combinatorial indexing strategy provided herein such that cells can be processed in bulk and single cell outputs can be demultiplexed in silico. To be done. In some embodiments, the methods provided herein utilize the incorporation of transposase-based adapters, which results in increased efficacy and much higher alignment rates than exiting the method. The use of transposase to add one of the two sequencing adapters allows for much more efficient library construction with less noise reading, thus using the single cell single well method 10. Alignment rates of approximately 60% (similar to the bulk cell strategy) are produced when compared to -30%. This results in more useful sequence reads and a dramatic cost savings for the sequencing portion of the assay. The use of a single cell combinatorial indexing strategy to generate a single cell bisulfite sequencing library is demonstrated on a mixture of human and mouse cells with minimal collision rates. Also, successful deconvolution of a mixture of three human cell types has been demonstrated to achieve cell type assignment using publicly available data.

Definitions As used herein, the terms "organism", "subject" are used interchangeably and refer to animals and plants. Examples of animals are mammals such as humans.

As used herein, the term "cell type" is intended to identify a cell based on morphology, phenotype, developmental origin, or other known or discernible distinctive cellular characteristic. .. A variety of different cell types can be obtained from a single organism (or organism of the same species). Exemplary cell types include bladder, pancreatic epithelium, pancreatic α, pancreatic β, pancreatic endothelium, bone marrow lymphoblasts, bone marrow B lymphoblastoma, bone marrow macrophages, bone marrow erythroblasts, bone marrow dendritic cells, bone marrow adipocytes, bone marrow. Bone cells, bone marrow chondrocytes, promyeloblast, myelomegaloblasts, bladder, brain B lymphocytes, glial cells, neurons, brain astrocytes, neuroectodermal, brain macrophages, brain microglia, brain Epithelium, cortical neuron, brain fibroblast, breast epithelium, colon epithelium, colon B lymphocyte, breast epithelium, breast myoepithelium, breast fibroblast, colon enterocyte, cervical epithelium, ovarian epithelium, ovarian fibroblast, Ductal epithelium, tongue epithelium, tonsil dendritic, tonsil B lymphocyte, peripheral blood lymphoblast, peripheral blood T lymphoblast, peripheral blood cutaneous T lymphocyte, peripheral blood natural killer, peripheral blood B lymphoblast, Peripheral blood monocytes, peripheral blood myeloblasts, peripheral blood monoblasts, peripheral blood promyelocytes, peripheral blood macrophages, peripheral blood basophils, liver endothelium, liver obesity, liver epithelium, liver B lymphocytes, spleen endothelium, Splenic epithelium, splenic B lymphocyte, hepatocyte, liver alexander, liver fibroblast, lung epithelium, bronchial epithelium, lung fibroblast, lung B lymphocyte, lung Schwann, lung squamous epithelium, lung macrophage, lung osteoblast, Neuroendocrine, alveoli, gastric epithelium, and gastric fibroblasts include, but are not limited to.

As used herein, the term "tissue" is intended to mean the collection or aggregation of cells that work together to carry out one or more specific functions in an organism. The cells may optionally be morphologically similar. Exemplary tissues include eyes, muscles, skin, tendons, veins, arteries, blood, heart, spleen, lymph nodes, bone, bone marrow, lungs, bronchi, trachea, intestine, small intestine, large intestine, colon, rectum, salivary gland, Tongue, gallbladder, appendix, liver, pancreas, brain, stomach, skin, kidney, ureter, bladder, urethra, gonad, testis, ovary, uterus, fallopian tube, thymus, pituitary gland, thyroid, adrenal gland, or parathyroid gland However, the present invention is not limited to these. The tissue can be from any of various organs of humans or other organisms. The tissue can be healthy tissue or unhealthy tissue. Examples of unhealthy tissues include various malignancies with abnormal methylation, such as lung, breast, colorectal, prostate, nasopharyngeal, stomach, testis, skin, nervous system, bone, ovary, liver, blood tissue. , But not limited to malignant tumors in the pancreas, uterus, kidneys, lymphoid tissues and the like. Malignant tumors can be of various histological subtypes, eg, carcinoma, adenocarcinoma, sarcoma, fibroadenocarcinoma, neuroendocrine, or undifferentiated.

As used herein, the term "compartment" is intended to mean a range or volume that separates or isolates something from another. Exemplary compartments include, but are not limited to, vials, tubes, wells, droplets, boluses, beads, containers, surface features, or ranges or volumes separated by physical forces such as fluid flow, magnetic forces, electric currents. Not limited. In one embodiment, the compartments are the wells of a multi-well plate such as a 96-well plate or a 384-well plate.

As used herein, "transposome complex" refers to a nucleic acid that contains an integrative enzyme and an integrative recognition site. A "transposome complex" is a functional complex formed by a transposase and a transposase recognition site capable of catalyzing a rearrangement reaction (see, for example, Gunderson et al., WO 2016/130704). Examples of incorporated enzymes include, but are not limited to, integrase or transposase and the like. Examples of integrated recognition sites include, but are not limited to, transposase recognition sites.

As used herein, the term "nucleic acid" is intended to be consistent with its use in the art and includes naturally occurring nucleic acids or functional analogs thereof. Particularly useful functional analogs can hybridize to nucleic acids in a sequence specific manner, or can be used as templates for the replication of specific nucleotide sequences. Naturally occurring nucleic acids generally have a backbone that contains phosphodiester bonds. The analog structure can have alternative backbone bonds, including any of a variety known in the art. Naturally occurring nucleic acids generally have a deoxyribose sugar (eg, found in deoxyribonucleic acid (DNA)) or a ribose sugar (eg, found in ribonucleic acid (RNA)). The nucleic acid can include any of the various analogs of these sugar moieties known in the art. Nucleic acids can include natural or unnatural bases. In this regard, the natural deoxyribonucleic acid can have one or more bases selected from the group consisting of adenine, thymine, cytosine or guanine, the ribonucleic acid being selected from the group consisting of uracil, adenine, cytosine or guanine. It may have one or more bases as described. Useful non-natural bases that can be included in nucleic acids are known in the art. Examples of non-natural bases include locked nucleic acids (LNA) and bridging nucleic acids (BNA). LNA and BNA bases can be incorporated into DNA oligonucleotides to increase oligonucleotide hybridization strength and specificity. LNA and BNA bases and the use of such bases are known and routine to those of skill in the art.

As used herein, the term "target", when used with reference to a nucleic acid, is intended as the semantic identifier of the nucleic acid in the context of the methods or compositions presented herein, or otherwise. It does not necessarily limit the composition or function of the nucleic acid beyond what is explicitly indicated. The target nucleic acid can be essentially any nucleic acid of known or unknown sequence. It can be, for example, a fragment of genomic DNA or cDNA. Sequencing can result in the determination of the sequence of all or part of a target molecule. The target can be derived from a primary nucleic acid sample such as a nucleus. In one embodiment, the target can be processed into a template suitable for amplification by placing a universal sequence at the end of each target fragment. Targets can also be obtained from the primary RNA sample by reverse transcription into cDNA.

As used herein, the term "generic", when used to describe a nucleotide sequence, refers to a region of sequence common to two or more nucleic acid molecules, which molecule also has regions of sequence that differ from each other. Point to. A universal sequence that is present in different members of a collection of molecules can be used to capture a plurality of different nucleic acids using a population of universal capture nucleic acids, e. Can be enabled. Non-limiting examples of universal capture sequences include sequences that are the same or complementary to the P5 and P7 primers. Similarly, a universal sequence that is present in a different member of a collection of molecules uses a portion of the universal sequence, for example, a population of universal primers that are complementary to a universal anchor sequence, to replicate or amplify multiple different nucleic acids. May enable. Thus, the capture oligonucleotide or universal primer comprises a sequence that can specifically hybridize to the universal sequence.

The terms "P5" and "P7" may be used when referring to amplification primers, such as capture oligonucleotides. The terms "P5'" (P5' primer) and "P7'" (P7' primer) refer to the complements of P5 and P7, respectively. It will be appreciated that any suitable amplification primer can be used in the methods presented herein, and the use of P5 and P7 is an exemplary embodiment only. The use of amplification primers such as P5 and P7 on flow cells is described in WO2007/010251, WO2006/064199, WO2005/065814, WO2015/106941. It is known in the art, as exemplified by the disclosures of the pamphlet, WO 1998/044151 and WO 2000/018957. For example, any suitable forward amplification primer, whether immobilized or in solution, is useful in the methods presented herein for hybridization to complementary sequences and amplification of sequences. possible. Similarly, any suitable reverse amplification primer, whether immobilized or in solution, is useful in the methods provided herein for hybridization to complementary sequences and amplification of sequences. Can be Those skilled in the art will understand how to design and use suitable primer sequences for the capture and/or amplification of nucleic acids as presented herein.

As used herein, the term "primer" and its derivatives generally refer to any nucleic acid capable of hybridizing to a target sequence of interest. Typically, the primer functions as a substrate whose nucleotides can be polymerized by a polymerase, but in some embodiments, the primer is incorporated into a synthesized nucleic acid strand and another primer hybridizes to the synthetic nucleic acid. It may provide a site for prime synthesis of a new strand that is complementary to the molecule. Primers can include any combination of nucleotides or analogs thereof. In some embodiments, the primer is a single stranded oligonucleotide or polynucleotide. The terms "polynucleotide" and "oligonucleotide" are used interchangeably herein to refer to polymeric forms of nucleotides of any length, including ribonucleotides, deoxyribonucleotides, their analogs, or their It may include a mixture. The term includes, as equivalents, either DNA or RNA analogs made from nucleotide analogs and is applicable to single-stranded (eg, sense or antisense) and double-stranded polynucleotides. Should be understood. The term as used herein also includes cDNA, which is complementary or copy DNA produced from an RNA template, for example by the action of reverse transcriptase. This term refers only to the primary structure of the molecule. Thus, the term includes triple-stranded, double-stranded and single-stranded deoxyribonucleic acid (“DNA”), as well as triple-stranded, double-stranded and single-stranded ribonucleic acid (“RNA”).

As used herein, the term "adapter" and its derivatives (eg, universal adapter) generally refers to any linear oligonucleotide that can be linked to the nucleic acid molecules of the present disclosure. In some embodiments, the adapter is not substantially complementary to the 3'or 5'ends of any target sequence present in the sample. In some embodiments, suitable adapter lengths range from about 10-100 nucleotides, about 12-60 nucleotides and about 15-50 nucleotides in length. In general, the adapter can include any combination of nucleotides and/or nucleic acids. In some aspects, the adapter can include one or more cleavable groups at one or more positions. In another aspect, the adapter can include a sequence that is substantially identical or substantially complementary to at least a portion of a primer, eg, a universal primer. In some embodiments, the adapter can include a barcode or tag to aid downstream error correction, identification, or ordering. The terms "adaptor" and "adapter" are used interchangeably.

As used herein, the term "each", when used in reference to a collection of items, is intended to identify individual items within the collection, but the context clearly indicates otherwise. Unless it does, it does not necessarily refer to every item in the collection.

As used herein, the term "transport" refers to the movement of molecules through a fluid. The term may include passive transport, such as migration of molecules along their concentration gradient (eg, passive diffusion). The term can also include active transport, where molecules can move along or against their concentration gradient. Thus, transport can include applying energy to move one or more molecules in a desired direction or to a desired location, such as an amplification site.

As used herein, "amplify", "amplification" or "amplification reaction" and their derivatives generally mean that at least a portion of a nucleic acid molecule is replicated or copied into at least one additional nucleic acid molecule. Refers to any action or process. The additional nucleic acid molecule optionally comprises a sequence that is substantially identical or substantially complementary to at least some portion of the template nucleic acid molecule. Template nucleic acid molecules can be single-stranded or double-stranded, and additional nucleic acid molecules can be single-stranded or double-stranded independently. Amplification optionally involves linear or exponential replication of the nucleic acid molecule. In some embodiments, such amplification can be performed using isothermal conditions, and in other embodiments, such amplification can include thermocycling. In some embodiments, the amplification is a multiplex amplification that involves the simultaneous amplification of multiple target sequences in a single amplification reaction. In some embodiments, "amplifying" comprises amplifying at least some portions of DNA and RNA based nucleic acids, alone or in combination. The amplification reaction can include any of the amplification processes known to those of skill in the art. In some embodiments, the amplification reaction comprises the polymerase chain reaction (PCR).

As used herein, "amplification conditions" and derivatives thereof generally refer to conditions suitable for amplifying one or more nucleic acid sequences. Such amplification can be linear or exponential. In some embodiments, amplification conditions can include isothermal conditions, or can include thermocycling conditions, or a combination of isothermal and thermocycling conditions. In some embodiments, suitable conditions for amplifying one or more nucleic acid sequences include polymerase chain reaction (PCR) conditions. Typically, amplification conditions are for amplifying nucleic acids such as one or more target sequences or for amplifying amplified target sequences linked to one or more adapters, eg, adapter-ligated amplification target sequences. Refers to sufficient reaction mixture. Generally, amplification conditions include a catalyst for amplification or nucleic acid synthesis, such as a polymerase; a primer with some complementarity to the nucleic acid to be amplified; and nucleotides to facilitate extension of the primer after hybridizing to the nucleic acid. , For example, deoxyribonucleotide triphosphate (dNTP). Amplification conditions may require hybridization or annealing of the primer to the nucleic acid, extension of the primer, and denaturing steps in which the extended primer is separated from the nucleic acid sequence undergoing amplification. Typically, the amplification conditions can, but need not, include thermocycling, and in some embodiments, the amplification conditions include multiple cycles in which the steps of annealing, extension, and separation are repeated. Typically, the amplification state will contain cations such as Mg ²⁺ or Mn ²⁺ and may also contain various modifiers of ionic strength.

As used herein, "reamplification" and its derivatives generally mean that at least a portion of the amplified nucleic acid molecule is from any suitable amplification process (in some embodiments, "secondary"). Referred to as amplification), thereby producing a reamplified nucleic acid molecule. The secondary amplification need not be the same as the original amplification process by which the amplified nucleic acid molecule was produced, nor that the amplified nucleic acid molecule is exactly the same or is completely complementary to the amplified nucleic acid molecule. Nonetheless, it is only necessary that the reamplified nucleic acid molecule comprises at least part of the amplified nucleic acid molecule or its complement. For example, reamplification can include different amplification conditions and/or the use of different primers, including different target-specific primers than the primary amplification.

As used herein, the term “polymerase chain reaction” (“PCR”) refers to the method of Mullis US Pat. Nos. 4,683,195 and 4,683,202, These describe methods for increasing the concentration of a segment of a polynucleotide of interest in a mixture of genomic DNA without cloning or purification. This process for amplifying a polynucleotide of interest involves introducing a large excess of two oligonucleotide primers into a DNA mixture containing the desired polynucleotide of interest, followed by a series of thermal cycles in the presence of DNA polymerase. It consists of The two primers are complementary to each strand of the double-stranded polynucleotide of interest. The mixture is first denatured at higher temperature and then the primer is annealed to the complementary sequence within the polynucleotide molecule of interest. After annealing, the primers are extended with a polymerase to form new pairs of complementary strands. The steps of denaturation, primer annealing and polymerase extension can be repeated many times (called thermocycling) to obtain high concentrations of the amplified segment of the desired polynucleotide of interest. The length of the amplified segment (amplicon) of the desired polynucleotide of interest is determined by the relative position of the primers with respect to each other, and thus this length is a controllable parameter. By repeating this process, the method is referred to as the "polymerase chain reaction" (hereinafter "PCR"). Since the desired amplified segment of the polynucleotide of interest will be the predominant (in terms of concentration) nucleic acid sequence in the mixture, they are said to be "PCR amplified." In a modification to the above method, the target nucleic acid molecule can be PCR amplified using multiple different primer pairs, optionally one or more primer pairs per target nucleic acid molecule of interest, thereby providing a multiplex PCR reaction. Form.

As defined herein, "multiplex amplification" refers to the selective and non-random amplification of two or more target sequences in a sample using at least one target-specific primer. In some embodiments, multiplex amplification is performed such that some or all of the target sequences are amplified in a single reaction vessel. The “plexi” or “plex” of a given multiplex amplification generally refers to the number of different target-specific sequences amplified during that single multiplex amplification. In some embodiments, the plexi can be about 12, 24, 48, 96, 192, 384, 768, 1536, 3072, 6144 or more. Several different methodologies, such as gel electrophoresis followed by densitometry, bioanalyzer quantification, or quantitative PCR, hybridization with labeled probes; biotinylated primer incorporation, followed by avidin-enzyme conjugate detection; It is also possible to detect the amplified target sequence (by incorporation of 32 P-labeled deoxynucleotide triphosphate into the amplified target sequence).

As used herein, "amplified target sequence" and its derivatives generally refer to nucleic acid produced by an amplified target sequence using target-specific primers and the methods provided herein. Refers to an array. The amplified target sequence can be either the same sense (ie, plus strand) or antisense (ie, minus strand) with respect to the target sequence.

As used herein, the terms “link”, “link” and their derivatives generally refer to covalently linking two or more molecules together, eg, two or more. Refers to the process for covalently linking nucleic acid molecules to each other. In some embodiments, linking comprises linking a nick between adjacent nucleotides of a nucleic acid. In some embodiments, the ligation comprises forming a covalent bond between the end of the first nucleic acid molecule and the end of the second nucleic acid molecule. In some embodiments, the linking forms a covalent bond between the 5'phosphate group of one nucleic acid and the 3'hydroxyl group of a second nucleic acid, thereby forming a linked nucleic acid molecule. Can be included. In general, for the purposes of this disclosure, the amplified target sequence may be ligated to an adapter to produce an adapter ligated amplified target sequence.

As used herein, "ligase" and its derivatives generally refer to any agent capable of catalyzing the linking of two substrate molecules. In some embodiments, the ligase comprises an enzyme capable of catalyzing the nicking between adjacent nucleotides of a nucleic acid. In some embodiments, the ligase catalyzes the formation of a covalent bond between the 5'phosphate of one nucleic acid molecule and the 3'hydroxyl of another nucleic acid molecule, thereby forming a linked nucleic acid molecule. Including enzymes that can. Suitable ligases can include, but are not limited to, T4 DNA ligase, T4 RNA ligase, and E. coli DNA ligase.

As used herein, "linking conditions" and derivatives thereof generally refer to conditions suitable for linking two molecules together. In some embodiments, the ligation conditions are suitable to seal nicks or gaps between nucleic acids. The term nick or gap, as used herein, is consistent with its use in the art. Typically, the nicks or gaps can be linked in the presence of an enzyme (eg, ligase) at a suitable temperature and pH. In some embodiments, T4 DNA ligase is capable of binding nicks between nucleic acids at temperatures of about 70-72°C.

The term “flow cell” as used herein refers to a chamber containing a solid surface through which one or more fluid reagents can flow. Examples of flow cells and associated fluidic systems and detection platforms that can be readily used in the methods of the present disclosure are incorporated herein by reference, eg, Bentley et al. , Nature 456:53-59 (2008), WO 04/018497; US Pat. No. 7,057,026; WO 91/06678; WO 07/123744; US US Pat. No. 7,329,492; US Pat. No. 7,211,414; US Pat. No. 7,315,019; US Pat. No. 7,405,281; and US patents. It is described in Japanese Patent Application Publication No. 2008/01082.

As used herein, the term "amplicon," when used in reference to a nucleic acid, means a product that copies a nucleic acid, which product is the same as or complementary to at least a portion of the nucleotide sequence of the nucleic acid. Having a nucleotide sequence that is targeted. An amplicon is any of various amplification methods that use a nucleic acid or its amplicon as a template, including, for example, polymerase extension, polymerase chain reaction (PCR), rolling circle amplification (RCA), ligation extension, or ligation chain reaction. Can be produced by An amplicon can be a nucleic acid molecule that has a single copy of a particular nucleotide sequence (eg, a PCR product) or multiple copies of a nucleotide sequence (eg, a concatemer product of RCA). The first amplicon of the target nucleic acid is typically a complementary copy. Subsequent amplicons are copies made from the target nucleic acid or from the first amplicon after the production of the first amplicon. The subsequent amplicon can have a sequence that is substantially complementary to, or is substantially identical to, the target nucleic acid.

As used herein, the term "amplification site" refers to a site in or on an array where one or more amplicons can be produced. The amplification site can be further configured to include, retain, or attach to at least one amplicon produced at that site.

As used herein, the term "array" refers to a population of sites that can be distinguished from each other according to their relative position. Different molecules at different sites of the array can be distinguished from each other according to the position of the sites in the array. Individual sites in the array may contain one or more molecules of a particular type. For example, a site may contain a single target nucleic acid molecule having a particular sequence, or a site may contain several nucleic acid molecules having the same sequence (and/or its complementary sequence). The sites of the array can be different features located on the same substrate. Exemplary features include, but are not limited to, wells within the substrate, beads (or other particles) within or on the substrate, protrusions from the substrate, ridges on the substrate, or channels within the substrate. .. The sites of the array can be separate substrates, each with a different molecule. Different molecules attached to separate substrates can be identified according to the position of the substrate on the surface with which the substrate is associated, or according to the position of the substrate in a liquid or gel. Exemplary arrays in which the discrete substrates are located on the surface include, but are not limited to, those with beads in the wells.

As used herein, the term "volume", when used in reference to a site and nucleic acid material, means the maximum amount of nucleic acid material that can occupy the site. For example, the term can refer to the total number of nucleic acid molecules that can occupy a site in a particular state. Other measures can be used as well, including, for example, the total mass of nucleic acid material that can occupy a site in a particular state, or the total number of copies of a particular nucleotide sequence. Typically, the volume of the site for the target nucleic acid is substantially equivalent to the volume of the site for the amplicon of the target nucleic acid.

As used herein, the term "capture agent" refers to a material, chemical, molecule, or portion thereof that is capable of attaching, retaining or binding to a target molecule (eg, target nucleic acid). Exemplary capture agents include a capture nucleic acid that is complementary to at least a portion of the target nucleic acid (also referred to herein as a capture oligonucleotide), a receptor capable of binding to the target nucleic acid (or a binding moiety attached thereto). Can form a covalent bond with a member of a body-ligand binding pair (eg, avidin, streptavidin, biotin, lectin, carbohydrate, nucleic acid binding protein, epitope, antibody, etc.) or a target nucleic acid (or binding moiety attached thereto). These include, but are not limited to, possible chemical reagents.

As used herein, the term "clonal population" refers to a population of nucleic acids that is homogenous with respect to a particular nucleotide sequence. Homogeneous sequences are typically at least 10 nucleotides in length, but can be longer, including at least 50, 100, 250, 500 or 1000 nucleotides in length. A clonal population can be derived from a single target nucleic acid or template nucleic acid. Typically, all of the nucleic acids in a clonal population will have the same nucleotide sequence. It is understood that a small number of mutations can occur in the clonal population (eg due to amplification artifacts) without departing from clonality.

As used herein, “providing” in the context of a composition, article, nucleic acid, or nucleus refers to making a composition, article, nucleic acid, or nucleus, composition, article, nucleic acid, or nucleus. Or otherwise obtaining the compound, composition, article, article, or nucleus.

The term "and/or" means one or all of the listed elements or a combination of any two or more of the listed elements.

The words "preferred" and "preferably" refer to embodiments of the invention that may afford certain benefits, under certain circumstances. However, under the same or different circumstances, other embodiments may be preferred. Furthermore, the description of one or more preferred embodiments does not indicate that another embodiment is not useful, and is not intended to exclude another embodiment from the scope of the present invention.

The term "comprising" and variations thereof do not have a limiting meaning where these terms appear in the description and claims.

Embodiments described herein in terms such as "comprises" or "comprising" are everywhere else described by the terms "consisting of" and/or "consisting essentially of." It is understood that similar embodiments are also provided.

Unless otherwise specified, "a," "an," "the," and "at least one" are used interchangeably and mean one or more.

Further, in the present specification, the description of the numerical range by the end point includes all the numbers included in the range (for example, 1 to 5 are 1, 1.5, 2, 2.75, 3, 3). .80, 4, 5, etc.).

For any method disclosed herein, including separate steps, the steps may occur in any feasible order. Further, if desired, any combination of two or more steps may be performed simultaneously.

Throughout this specification, reference to "an embodiment," "an embodiment," "a particular embodiment," "some embodiments," or the like refers to a particular feature described in connection with that embodiment. , Composition, composition, or property is included in at least one embodiment of the present disclosure. Thus, the appearances of such phrases in various places throughout the specification are not necessarily all referring to the same embodiment of the disclosure. Furthermore, the particular features, configurations, compositions, or characteristics may be combined in any suitable manner in one or more embodiments.

The following detailed description of specific embodiments of the present disclosure can be best understood when read in conjunction with the following drawings.

FIG. 6 shows a general block diagram of a general exemplary method for single cell combination index addition according to the present disclosure. FIG. 1 shows a schematic diagram of one embodiment of a method for single cell combinatorial indexing generally shown in FIG. Figure 3 shows a schematic of an exemplary embodiment of the fragment-adaptor molecule after linear amplification. Figure 3 shows a schematic of an exemplary embodiment of a fragment-adapter molecule after addition of a universal adapter.

The schematic is not necessarily isometric. Like numbers used in the figures refer to like components, steps, and the like. It will be appreciated, however, that the use of numbers to refer to components in a given figure is not intended to limit components in other figures that are labeled with the same number. Furthermore, the use of different numbers to refer to components is not intended to indicate that different numbered components may not be the same or similar to other numbered components.

The methods provided herein include providing isolated nuclei from multiple cells (Figure 1, block 12). The cell may be derived from any organism, and may be derived from any cell type or any tissue of the organism. The method may further comprise dissociating the cells (Figure 2, block i) and/or isolating the nucleus (Figure 2, block ii). Methods for isolating nuclei from cells are well known and routine to those of skill in the art. The number of nuclei can be at least 2. The upper limit depends on the practical limits of the equipment (eg, multiwell plate) used in the other steps of the methods described herein. For example, in one embodiment, the number of nuclei is 1,000,000 or less, 100,000,000 or less, 10,000,000 or less, 1,000,000 or less, 10,000 or less, or 1, 000 or less. Those skilled in the art will recognize that the nucleic acid molecule in each nucleus represents the entire genetic complement of the organism and is a genomic DNA molecule containing both intron and exon sequences and non-coding regulatory sequences such as promoter and enhancer sequences. Will.

In one embodiment, the nucleus comprises nucleosomes bound to genomic DNA. Such nuclei may be useful in methods that do not sequence the entire genome of the cell, such as sciATAC-seq. In another embodiment, the isolated nuclei are subjected to conditions that deplete the nucleosomal nuclei to produce nucleosomal depleted nuclei (FIG. 1, block 13, and FIG. 2, block ii). Such nuclei may be useful in methods aimed at determining the total genomic DNA sequence of a cell. In one embodiment, the conditions used for nucleosome depletion maintain the integrity of the isolated nucleus. Methods for generating nucleosome-depleted nuclei are known to those of skill in the art (see, eg, Vitak et al., 2017, Nature Methods, 14(3):302-308). In one embodiment, the conditions are chemical treatments, including treatment with chaotropic agents that can disrupt nucleic acid-protein interactions. Examples of useful chaotropic agents include, but are not limited to, lithium diiodosalicylate. In another embodiment, the conditions are chemical treatments, including treatment with detergents that can disrupt nucleic acid-protein interactions. An example of a useful detergent includes, but is not limited to, sodium dodecyl sulfate (SDS). In some embodiments, when a detergent such as SDS is used, the cells from which the nuclei are isolated are treated with a crosslinker prior to isolation. Useful examples of cross-linking agents include, but are not limited to, formaldehyde.

The methods provided herein include partitioning a subset of nuclei (eg, nucleosome depleted nuclei) into a first plurality of compartments (FIG. 1, block 14 and FIG. 2, left side schematic). The number of nuclei present in the subset, and thus the number of nuclei present in each compartment, can be at least one. In one embodiment, the number of nuclei present in the subset is 2,000 or less. Methods for partitioning nuclei into subsets are well known and routine to those of skill in the art. An example includes, but is not limited to, fluorescence activated nuclear sorting (FANS).

Each compartment contains a transposome complex. The transposase complex, which is a transposase bound to a transposase recognition site, can insert the transposase recognition site into a target nucleic acid in the nucleus in a process sometimes called “tagmentation”. In some such insertion events, one strand of the transposase recognition site can be transferred to the target nucleic acid. Such chains are called "transport chains". In one embodiment, the transposome complex comprises a dimeric transposase with two subunits and two discontinuous transposon sequences. In another embodiment, the transposase comprises a dimeric transposase having two subunits and a continuous transposon sequence.

Some embodiments include an overactive Tn5 transposase and a Tn5-type transposase recognition site (Goryshin and Reznikoff, J. Biol. Chem., 273:7367 (1998)), or a Mu transposase that includes a MuA transposase and R1 and R2 terminal sequences. The use of recognition sites (Mizuchi, K., Cell, 35:785, 1983; Savirahti, H, et al., EMBO J., 14:4893, 1995) may be involved. The Tn5 mosaic end (ME) sequence can also be optimized and used by those skilled in the art.

Further examples of transposition systems that can be used in certain embodiments of the compositions and methods provided herein are Staphylococcus aureus Tn552 (Colegio et al., J. Bacteriol., 183:2384). -8, 2001; Kirby C et al., Mol. Microbiol., 43:173-86, 2002), Ty1 (Device & Boeke, Nucleic Acids Res., 22:3765-72, 1994 and International Publication No. 95/23875. No. Pamphlet), transposon Tn7 (Craig, NL, Science. 271:1512, 1996; Craig, NL, Review in: Curr Top Microbiol Immunol., 204:27-48, 1996), Tn/O and IS10 (Kleckner). N, et al., Curr Top Microbiol Immunol., 204:49-82, 1996), Mariner transposase (Lampe DJ, et al., EMBO J., 15:5470-9, 1996), Tc1 (Plasterk RH. , Curr. Topics Microbiol. Immunol., 204:125-43, 1996), P element (Gloor, GB, Methods Mol. Biol., 260:97-114, 2004), Tn3 (Ichikawa & Ohtsubo, J Bio. Chem. 265: 18829-32, 1990), bacterial insertion sequence (Ohtsubo & Sekine, Curr. Top. Microbiol. Immunol. 204:1-26, 1996), retrovirus (Brown, et al., Proc Natl Acad Sci USA). , 86:2525-9, 1989), and yeast retrotransposons (Boeke & Centers, Annu Rev Microbiol. 43:403-34, 1989). Further examples include engineered versions of IS5, Tn10, Tn903, IS911, and transposase family enzymes (Zhang et al., (2009) PLoS Genet. 5:e1000689.Epub 2009 Oct 16; Wilson C. et al( 2007) J. Microbiol. Methods 71:332-5).

Another example of an integrase that can be used in the methods and compositions provided herein is a retroviral integrase such as an integrase from HIV-1, HIV-2, SIV, PFV-1, RSV. And the integrase recognition sequences of such retroviral integrases.

Transposon sequences useful in the methods and compositions provided herein are disclosed in US Patent Application Publication No. 2012/0208705, US Patent Application Publication No. 2012/0208724, and International Patent Application Publication No. 2012/061832. It is provided in the issue brochure. In some embodiments, the transposon sequence comprises a first transposase recognition site, a second transposase recognition site, and an index that lies between these two transposase recognition sites.

Some transposome complexes useful herein include transposases having two transposon sequences. In some such embodiments, the two transposon sequences are not linked to each other, in other words the transposon sequences are discontinuous with each other. Examples of such transposomes are known in the art (see, eg, US Patent Application Publication No. 2010/0120098).

In some embodiments, the transposome complex comprises a transposon sequence nucleic acid that binds to two transposase subunits to form a "loop complex" or "loop transposome." In one example, the transposome comprises dimeric transposase and transposon sequences. The looped complex can ensure that the transposon is inserted into the target DNA without fragmenting the target DNA while maintaining the ordering information of the original target DNA. As will be appreciated, the looped structure can insert a desired nucleic acid sequence, such as an index, into the target nucleic acid while maintaining the physical connectivity of the target nucleic acid. In some embodiments, the transposon sequences of the looped transposome complex can include fragmentation sites such that the transposon sequences can be fragmented to produce a transposome complex that contains two transposon sequences. Such transposome complexes are useful to ensure that the flanking target DNA fragments into which the transposon is inserted receive a coding combination that can be unambiguously assembled in later steps of the assay.

The transposome complex also contains at least one index sequence, also called the transposase index. The index sequence exists as part of the transposon sequence. In one embodiment, the index sequence may be on a transport strand that is the strand of the transposase recognition site that is transported to the target nucleic acid. Index sequences, also called tags or barcodes, are useful as marker features in the compartment where a particular target nucleic acid was present. The index sequence of the transposome complex differs from compartment to compartment. Therefore, in this embodiment, the index is a nucleic acid sequence tag attached to each of the target nucleic acids present in a particular compartment, the presence of which indicates the compartment in which the population of nuclei was present at this stage of the method, or Or used to identify.

Index sequences are up to 20 nucleotides in length, eg 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19. , 20. The 4-nucleotide tag offers the possibility to multiplex 256 samples on the same array and the 6-base tag allows 4096 samples to be processed on the same array.

In one embodiment, the transport strand can also include a universal sequence, a first sequencing primer sequence, or a combination thereof. Universal sequences and sequencing primer sequences are described herein. Thus, in some embodiments in which the transfer strand is transferred to the target nucleic acid, the target nucleic acid comprises a transposase index and also comprises a universal sequence, a first sequencing primer sequence, or a combination thereof.

In one embodiment, the transport chain cytosine nucleotides are methylated. In another embodiment, the nucleotides of the transport strand do not include cytosine. Any sequence present on the transport chain, including such a transport chain and a transposase index sequence, a universal sequence, and/or a first sequencing primer sequence, can be referred to as cytosine depleted. The use of cytosine-depleted nucleotide sequences in the transposome complex has no significant effect on transposase efficiency.

The method also includes generating indexed kernels (FIG. 1, block 15, and FIG. 2, block iii). In one embodiment, generating the indexed nuclei comprises fragmenting nucleic acids present in a subset of nucleosome depleted nuclei (eg, nucleic acids present in each compartment) into multiple nucleic acid fragments. In one embodiment, fragmentation of nucleic acids is accomplished by using the fragmentation sites present in the nucleic acid. Typically, the fragmentation site is introduced into the target nucleic acid by using the transposome complex. For example, the looped transposome complex can include a fragmentation site. Fragmentation sites can be used to break physical rather than informative relationships between index sequences inserted into a target nucleic acid. Cleavage may be by biochemical, chemical or other means. In some embodiments, the fragmentation site can include nucleotides or nucleotide sequences that can be fragmented by various means. Examples of fragmentation sites are a restriction endonuclease site, at least one ribonucleotide cleavable by RNAse, a nucleotide analog cleavable in the presence of certain chemical agents, cleavable by treatment with periodate. Examples include, but are not limited to, diol bonds, disulfide groups that are cleavable by chemical reducing agents, cleavable moieties that can be subject to photochemical cleavage, and peptides that are cleavable by peptidase enzymes or other suitable means (eg, US See Patent Application Publication No. 2012/0208705, US Patent Application Publication No. 2012/0208724, and International Publication No. WO 2012/061832 pamphlet). The result of the fragmentation is a population of indexed nuclei, each nucleus containing a nucleic acid fragment, wherein the nucleic acid fragment comprises, in at least one strand, an index sequence that represents a particular compartment.

Indexed kernels from multiple compartments can be combined (FIG. 1, block 16, and FIG. 2, left-hand schematic). For example, indexed nuclei from 2 to 96 compartments (if 96 well plates are used) or 2 to 384 compartments (if 384 well plates are used) are combined. A subset of these combined indexed nuclei, referred to herein as pooled indexed nuclei, is then distributed to the second plurality of partitions. The number of nuclei present in the subset, and thus in each compartment, is based in part on the desire to reduce index collisions, which leads to the same transposase index ending in the same compartment at this step of the method. It is the existence of two nuclei. In this embodiment, the number of nuclei present in the subset is between 2 and 30, for example 2,3,4,5,6,7,8,9,10,11,12,13,14,15,16. , 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30. In one embodiment, the number of nuclei present in the subset is 20-24, eg 22. Methods for partitioning nuclei into subsets are well known and routine to those of skill in the art. An example includes, but is not limited to, fluorescence activated nuclear sorting (FANS).

The distributed indexed nuclei are processed to identify methylated nucleotides (FIG. 1, block 17, and FIG. 2, block iv). Methylation of sites such as CpG dinucleotide sequences can be measured using any of the various techniques used in the art for analysis of such sites. One useful method is the identification of methylated CpG dinucleotide sequences. Identification of methylated CpG dinucleotide sequences was determined using techniques based on cytosine conversion, which rely on methylation state-dependent chemical modification of CpG sequences within isolated genomic DNA or fragments thereof, followed by DNA sequence analysis. To be done. Chemical reagents capable of distinguishing between methylated and unmethylated CpG dinucleotide sequences include hydrazine, which cleaves nucleic acids, and bisulfite. Alkaline hydrolysis followed by bisulfite treatment specifically converts unmethylated cytosine to uracil, and was reported by Olek A. et al. , 1996, Nucleic Acids Res. 24:5064-6 or Frommer et al. , 1992, Proc. Natl. Acad. Sci. 5-methylcytosine remains unmodified as described by USA 89:1827-1831. The bisulfite treated DNA can subsequently be analyzed by molecular techniques such as PCR amplification, sequencing, and detection including oligonucleotide hybridization (eg, using nucleic acid microarrays). In one embodiment, the indexed nuclei within each compartment are exposed to conditions for bisulfite treatment. Bisulfite treatment of nucleic acids is well known and routine to those of skill in the art. In one embodiment, bisulfite treatment converts the unmethylated cytosine residues of CpG dinucleotides to uracil residues leaving the 5-methylcytosine residues unchanged. Bisulfite treatment results in bisulfite treated nucleic acid fragments.

After generation of the bisulfite treated nucleic acid fragment, the fragment is modified to include additional nucleotides at one or both ends (Figure 1, block 18, and Figure 2, blocks v and vi). In one embodiment, the modification comprises subjecting the bisulfite treated nucleic acid fragment to linear amplification using multiple primers. Each primer contains at least two regions; a universal nucleotide sequence at the 5'end and a random nucleotide sequence at the 3'end. The universal nucleotide sequence is the same in each primer and, in one embodiment, comprises a second sequencing primer sequence (also referred to as the read 2 primer in Figure 2) (block vii). Regions of random nucleotide sequences are used such that there should be at least one primer that is complementary to all sequences in the bisulfite treated nucleic acid fragment. The number of random nucleotides that can be used to increase the probability of full coverage to the desired level can be determined using routine methods, and can range from 6 to 12 random nucleotides, eg, 9 random nucleotides. It can be a nucleotide. In one embodiment, the number of cycles is limited to 10 cycles or less, such as 9 cycles, 8 cycles, 7 cycles, 6 cycles, 5 cycles, 4 cycles, 3 cycles, 2 cycles, or 1 cycle. The result of linear amplification is the amplified fragment-adaptor molecule. Examples of fragment-adaptor molecules are shown in FIG. Fragment-adapter molecule 30 comprises nucleotides from the transferred strand of transposome complexes 31 and 32, including transposase index and universal sequences that can be used for amplification and/or sequencing. The fragment-adapter molecule also comprises nucleotides from the nuclear 33 genomic DNA, a region of random nucleotide sequence 34, and universal nucleotide sequence 35.

After linear amplification, an exponential amplification reaction, such as PCR, is followed to further modify the ends of the fragment-adaptor molecule before immobilization and sequencing. This step results in the fragment-adapter molecule indexing by PCR (FIG. 1, block 19). The universal sequences 31, 32 and/or 35 present at the ends of the fragment-adapter molecule serve as primers and can be used for the attachment of universal anchor sequences which can be extended in amplification reactions. Two different primers are typically used. One primer hybridizes to the universal sequence at the 3'end of one strand of the fragment-adapter molecule and the second primer hybridizes to the universal sequence at the 3'end of the other strand of the fragment-adaptor molecule. .. Therefore, the anchor sequence of each primer may be different. Suitable primers can each include an additional universal sequence, such as a universal capture sequence, and another index sequence. Since each primer can contain an index, this step results in the addition of one or two index sequences, eg a second and optionally a third index. Fragment-adapter molecules with a second and optionally a third index are called dual-index fragment-adapter molecules. The second and third indexes can be inverse complements of each other, or the second and third indexes can have sequences that are not inverse complements of each other. This second index array and optionally the third index is unique to each compartment in which the distributed indexed nuclei were placed prior to treatment with sodium bisulfite. The result of this PCR amplification is a fragment or adapter molecule of multiple or library having a similar or identical composition to the fragment-adaptor molecule shown in Figure 2, block vii.

In another embodiment, the modification comprises subjecting the bisulfite treated nucleic acid fragment to conditions that result in the ligation of additional sequences to both ends of the fragment. In one embodiment, blunt end ligation can be used. In another embodiment, the fragment is, for example, Taq polymerase or Klenow exo having non-template-dependent terminal transferase activity that adds a single deoxynucleotide, such as deoxyadenosine (A), to the 3'end of the bisulfite treated nucleic acid fragment. It is prepared with a single overhanging nucleotide, depending on the activity of a particular type of DNA polymerase, such as a minus polymerase. Such enzymes can be used to add a single nucleotide "A" to the blunt-ended 3'end of each strand of the fragment. Thus, "A" can be added to the 3'end of each strand of the double stranded target fragment by reaction with Taq or Klenow exo-minase polymerase, while additional sequences added to each end of the fragment are added to the 2'end. It may include compatible "T" overhangs present on the 3'end of each region of the double-stranded nucleic acid. This end modification also prevents self-ligation of the nucleic acids, thus biasing the formation of bisulfite treated nucleic acid fragments flanking the sequence added in this embodiment.

Fragmentation of nucleic acid molecules by the methods described herein results in fragments with blunt ends and a heterogeneous mixture of 3'and 5'overhanging ends. Thus, fragment ends are repaired using methods or kits known in the art (eg, Lucigen DNA terminator end repair kit), eg, to generate optimal ends for insertion into the blunt site of a cloning vector. Is desirable. In certain embodiments, the fragment ends of the nucleic acid population are blunt ended. More specifically, the fragment ends are blunt-ended and phosphorylated. The phosphate moiety can be introduced via enzymatic treatment, eg, using a polynucleotide kinase.

In one embodiment, the bisulfite-treated nucleic acid fragment is initially labeled with identical universal adapters (also referred to as "mismatch adapters") at the 5'and 3'ends of the bisulfite-treated nucleic acid fragment. Are described in Gormley et al., US Pat. No. 7,741,463, and Bignell et al., US Pat. No. 8,053,192). It is processed by forming an adapter molecule. In one embodiment, the universal adapter contains all the sequences required for sequencing and comprises immobilizing the fragment-adapter molecule on the array. Since the nucleic acid to be sequenced is from a single cell, further amplification of the fragment-adapter molecule is useful to achieve a sufficient number of fragment-adapter molecules for sequencing.

In another embodiment, if the universal adapter does not contain all the sequences required for sequencing, a PCR step is used to further supplement the universal adapter present in each fragment-adapter molecule prior to immobilization and sequencing. It can be modified. For example, the initial primer extension reaction is performed using a universal anchor sequence complementary to the universal sequence present in the fragment-adapter molecule, where complementary to both strands of each individual fragment-adaptor molecule. An extension product is formed. Typically, PCR adds additional universal sequences, such as universal capture sequences, and another index sequence. Since each primer can contain an index, this step results in the addition of one or two index sequences, eg a second and optionally a third index, and a fragment-adapter molecule indexing by adapter ligation ( FIG. 1, block 19). The resulting fragment-adapter molecule is called dual-index fragment-adapter molecule.

A universal adapter was added by a single step method of ligating a universal adapter containing all the sequences required for sequencing, or by a two step method of ligating the universal adapter and then PCR amplifying to further modify the universal adapter. Afterwards, the final fragment-adapter molecule will contain a universal capture sequence, a second index sequence, and optionally a third index sequence. These indexes are similar to the second and third indexes described in the production of dual-index fragment-adapter by linear amplification. The second and third indexes can be anti-complementary to each other, or the second and third indexes can have sequences that are not anti-complementary to each other. These second and optional third index sequences are unique to each compartment in which the distributed indexed nuclei were placed prior to treatment with sodium bisulfite. As a result of adding universal adapters to both ends, a plurality of fragments-adaptor molecules or a library having a structure similar to or the same as the fragments-adaptor molecule 40 shown in FIG. 4 is obtained. Fragment-adapter molecule 40 comprises capture sequences 41 and 48, also referred to as 3'flow cell adapters (e.g. P5) and 5'flow cell adapters (e.g. P7'), respectively, and indices 42 and 47, such as i5 and i7. Fragment-adapter molecule 40 also includes nucleotides from the transport strand of transposome complex 43, including transposase index 44 and universal sequence 45 that can be used for amplification and/or sequencing. The fragment-adapter molecule also contains nucleotides derived from the nuclear 46 genomic DNA.

The resulting dual-index fragment-adapter molecules collectively provide a library of nucleic acids that can be immobilized and then sequenced. The term library refers to a collection of single cell derived fragments containing known universal sequences at their 3'and 5'ends.

After the bisulfite treated nucleic acid fragment has been modified to contain additional nucleotides, dual-index fragment-adapter molecules are selected for a given size range, such as 150-400 nucleotides in length, eg 150-300 nucleotides. Subject to conditions. The resulting dual-index fragment-adapter molecules can be pooled and optionally subjected to a cleaning process to increase the purity of the DNA molecule by removing at least a portion of the unincorporated universal adapter or primer. it can. Any suitable cleaning process can be used such as electrophoresis, size exclusion chromatography and the like. In some embodiments, solid phase reversibly immobilized paramagnetic beads can be used to separate desired DNA molecules from unattached universal adapters or primers and to select nucleic acids based on size. Solid-phase reversibly immobilized paramagnetic beads are available from Beckman Coulter (Agencourt AMPure XP), Thermofisher (MagJet), Omega Biotek (Mag-Bind), Promega Beads (Promega), and KapaBay (Promega), and KapaBay (Promega) and KapaBay (Promega). There is.

Multiple fragment-adapter molecules can be prepared for sequencing. After pooling the fragment-adapter molecules, they are immobilized and amplified before sequencing (Figure 1, block 20). Methods of attaching fragment-adapter molecules to substrates from one or more sources are known in the art. Similarly, methods for amplifying immobilized fragment-adaptor molecules include, but are not limited to, bridge amplification and kinetic exclusion. Methods for immobilization and amplification prior to sequencing are described, for example, by Bignell et al. (US Pat. No. 8,053,192), Gunderson et al. (International Publication No. 2016/130704 pamphlet), Shen et al. (U.S. Pat. No. 8,895,249), and Pipenburg et al. (US Pat. No. 9,309,502).

Pooled samples can be immobilized in preparation for sequencing. Sequencing can be performed as an array of single molecules or can be amplified prior to sequencing. Amplification can be performed using one or more immobilized primers. Immobilized primers can be on a flat surface or on a pool of beads. The bead pool can be separated into emulsions with a single bead in each "compartment" of the emulsion. At a concentration of only one template per "compartment", only a single template is amplified on each bead.

The term "solid phase amplification" as used herein refers to on a solid support or to a solid support such that all or part of the amplification products are immobilized on the solid support as they are formed. Refers to any nucleic acid amplification reaction performed in association with a support. In particular, this term includes solid phase polymerase chain reaction (solid phase PCR) and solid phase isothermal amplification, except that one or both of the forward and reverse amplification primers are immobilized on a solid support. Thus, the reaction is similar to standard solution phase amplification. Solid phase PCR involves the formation of an emulsion in which one primer is immobilized on beads and the other is in free solution, and colony formation in a solid phase gel matrix in which one primer is immobilized on the surface and one is in free solution. Includes the system.

In some embodiments, the solid support comprises a patterned surface. "Patterned surface" refers to the placement of different regions in or on an exposed layer of a solid support. For example, one or more regions can be a feature where one or more amplification primers are present. Features can be separated by interstitial regions where amplification primers are not present. In some embodiments, the pattern can be an xy format of features in rows and columns. In some embodiments, the pattern can be a repeating array of features and/or gap regions. In some embodiments, the pattern can be a random arrangement of features and/or gap regions. Exemplary patterned surfaces that can be used in the methods and compositions described herein are US Pat. Nos. 8,778,848, 8,778,849, and No. 9,079,148, as well as U.S. Patent Application Publication No. 2014/0243324.

In some embodiments, the solid support comprises an array of wells or depressions on the surface. It can be manufactured as is commonly known in the art using a variety of techniques including, but not limited to, photolithography, stamping techniques, molding techniques, and microetching techniques. As will be appreciated by those in the art, the technique used will depend on the composition and shape of the array substrate.

Patterned surface features include patterned covalent gels (eg, poly(N-(5-azidoacetamidylpentyl)acrylamide-co-acrylamide) (PAZAM, eg, US Patent Publication No. 2013/ Wells on glass, silicon, plastic, or other suitable solid support (see, eg, 184796, WO 2016/066586, and WO 2015/002813) (eg, , Microwells or nanowells) This process creates a gel pad used for sequencing, which gel pad may be stable over sequencing runs with multiple cycles. Covalent attachment of polymers to the wells helps maintain the gel within the structured features throughout the life of the structured substrate during various uses, however, in many embodiments, the gel is covalently attached to the wells. For example, in some conditions, silane-free acrylamides (SFAs, such as US Pat. No. 8,563,477) may be used as the gel material.

In certain embodiments, the structured substrate patterns a solid support material with wells (eg, microwells or nanowells) and the patterned support with a gel material (eg, PAZAM, SFA, or azidoyzed SFA). ) Versions (chemically modified variants thereof such as azido-SFA) coated and gel coated supports are abraded, for example by chemical or mechanical polishing, thereby retaining the gel in the well but not the well. It can be made by removing or inactivating substantially all gel from the interstitial regions on the surface of the structured substrate in between. The primer nucleic acid can be attached to the gel material. The solution of fragment-adapter molecules can then be contacted with a polished substrate such that the individual fragment-adaptor molecules are seeded into individual wells through interaction with the primer attached to the gel material. However, the target nucleic acid will not occupy the interstitial region due to the absence or inactivity of the gel material. Amplification of fragment-adaptor molecules is limited to wells, as the absence or inactivity of the gel in the stromal region prevents outward migration of growing nucleic acid colonies. This process is conveniently manufacturable, scalable, and utilizes conventional micro or nano fabrication methods.

The present disclosure encompasses "solid phase" amplification methods in which only one amplification primer is immobilized (other primers are usually in free solution), but on a solid support, immobilized forward Preferably both a primer and a reverse primer are provided. In practice, the amplification process requires an excess of primers to maintain amplification, so "multiple" identical forward primers and/or "multiple" identical reverse primers immobilized on a solid support. Will exist. References to forward and reverse primers herein should be construed as including "plurality" of such primers unless the context dictates otherwise.

As will be appreciated by those in the art, any given amplification reaction will require at least one type of forward primer and at least one type of reverse primer specific for the template being amplified. However, in certain embodiments, the forward and reverse primers can include template-specific portions of the same sequence and can have exactly the same nucleotide sequence and structure (including any non-nucleotide modifications). In other words, it is possible to perform solid phase amplification using only one type of primer, and such single primer methods are included within the scope of the invention. Another embodiment may use forward and reverse primers that contain the same template-specific sequence, but differ in some other structural features. For example, one type of primer may contain non-nucleotide modifications that are not present in the other.

In all embodiments of the present disclosure, the primer for solid phase amplification is preferably immobilized by a single point covalent bond to a solid support at or near the 5'end of the primer and the template-specific portion of the primer is , Remains free to anneal to its cognate template and the 3′ hydroxyl group is free due to primer extension. Any suitable covalent means known in the art can be used for this. The attachment chemistry chosen will depend on the nature of the solid support, and any derivatization or functionalization applied to it. The primer itself can include moieties that can be non-nucleotide chemical modifications to facilitate attachment. In certain embodiments, the primer can include a sulfur-containing nucleophile such as phosphorothioate or thiophosphate at the 5'end. In the case of solid-supported polyacrylamide hydrogels, this nucleophile can bind to bromoacetamide groups present in the hydrogel. A more specific means of attaching the primer and template to the solid support is described in WO 05/0658814, as fully described by polymerized acrylamide and N-(5-bromoacetamidylpentyl)acrylamide. Via 5'phosphorothioate attachment to a hydrogel composed of (BRAPA).

Certain embodiments of the present disclosure are, for example, "functionalized" inert substrates or matrices by the application of layers or coatings of intermediate materials that include reactive groups that allow covalent attachment to biomolecules such as polynucleotides. A solid support composed of (eg, glass slides, polymer beads, etc.) may be utilized. Examples of such supports include, but are not limited to, polyacrylamide hydrogels supported on an inert substrate such as glass. In such embodiments, the biomolecule (eg, polynucleotide) may be directly covalently bound to the intermediate material (eg, hydrogel), but the intermediate material itself is non-attached to the matrix or matrix (eg, glass matrix). It may be covalently bound. The term "covalent bond to a solid support" should therefore be construed as encompassing this type of arrangement.

Pooled samples can be amplified on beads where each bead contains a forward and reverse amplification primer. In certain embodiments, a library of fragment-adapter molecules is prepared by solid phase amplification, more specifically solid phase isothermal amplification, US Patent Application Publication No. 2005/0100900, US Patent No. 7,115,400. It is used to prepare a clustered array of nucleic acid colonies similar to those described in the specification, WO 00/18957 and WO 98/44151. The terms “cluster” and “colony” are used interchangeably herein to refer to distinct sites on a solid support that are composed of multiple identical immobilized nucleic acid strands and multiple identical immobilized complementary nucleic acid strands. To be used. The term "clustered array" refers to an array formed from such clusters or colonies. In this context, the term "array" should not be understood as requiring an ordered arrangement of clusters.

The term "solid phase" or "surface" refers to a planar array in which primers are attached to a flat surface (eg, glass, silica or plastic microscope slides or similar flow cell devices); one or two primers are attached to beads. , Beads to which the beads are amplified; or an array of beads on the surface after the beads have been amplified.

Clustered arrays are either processes of thermocycling as described in WO 98/44151 or processes in which the temperature is kept constant and the cycles of extension and denaturation are carried out with the modification of reagents. It can be prepared using Such an isothermal amplification method is described in patent application number WO 02/46456 and US patent application WO 2008/0009420. This is particularly preferred because of the lower temperatures useful in isothermal processes.

Any of the amplification methodologies described herein or generally known in the art can be used with a universal or target-specific primer to amplify an immobilized DNA fragment. Will understand. Suitable methods for amplification include Polymerase Chain Reaction (PCR), Strand Displacement Amplification (SDA), Transcription Mediated Amplification (TMA) and US Pat. No. 8,003,033, which is incorporated herein by reference in its entirety. Nucleic acid sequence-based amplification (NASBA) as described in US Pat. No. 354,354, but is not limited thereto. The amplification methods described above can be used to amplify one or more nucleic acids of interest. For example, the immobilized DNA fragment can be amplified using PCR including multiplex PCR, SDA, TMA, NASBA and the like. In some embodiments, a primer specifically directed to the polynucleotide of interest is included in the amplification reaction.

Other suitable methods for amplification of polynucleotides include oligonucleotide extension and ligation, rolling circle amplification (RCA) (Lizardi et al., Nat. Genet. 19:225-232 (1998)) and oligonucleotide ligation assays. (OLA) (generally US Pat. Nos. 7,582,420, 5,185,243, 5,679,524 and 5,573,907. EP 0 320 308 B1; EP 0 336 731 B1; EP 0 439 182 B1; WO 90/01069 pamphlet; WO 89/12696 Pamphlet; as well as WO 89/09835 pamphlet) technology. It will be appreciated that these amplification methodologies can be designed to amplify immobilized DNA fragments. For example, in some embodiments, the amplification method can include a ligation probe amplification or an oligonucleotide ligation assay (OLA) reaction that includes a primer that is specifically directed to the nucleic acid of interest. In some embodiments, the amplification method can include a primer extension-ligation reaction that includes a primer that is specifically directed to the nucleic acid of interest. As a non-limiting example of primer extension and ligation primers that can be specifically designed to amplify a nucleic acid of interest, amplification is described in US Pat. Nos. 7,582,420 and 7,611,869. As exemplified by the specification, it may include primers used for the GoldenGate assay (Illumina, Inc., San Diego, CA).

Exemplary isothermal amplification methods that can be used in the methods of the present disclosure are described, for example, in Dean et al. , Proc. Natl. Acad. Sci. USA 99:5261-66 (2002), or Multiple Isolation Amplification (MDA), as exemplified by US Pat. No. 6,214,587, for example. Not limited to. Other non-PCR based methods that can be used in the present disclosure are described, for example, in Walker et al. , Molecular Methods for Virus Detection, Academic Press, Inc. 1995; U.S. Pat. Nos. 5,455,166 and 5,130,238; and Walker et al. , Nucl. Acids Res. 20:1691-96 (1992), or strand displacement amplification (SDA), or, for example, Lage et al. , Genome Res. 13:294-307 (2003). The isothermal amplification method can be used with strand-displacing Phi 29 polymerase or Bst DNA polymerase large fragments, 5'->3' exo for random primer amplification of genomic DNA. The use of these polymerases takes advantage of their high propensity and strand displacement activity. The high kinetic properties allow the polymerase to produce fragments of 10-20 kb in length. As noted above, smaller fragments can be produced under isothermal conditions using a polymerase with low propensity and strand displacement activity such as Klenow polymerase. Further description of amplification reactions, conditions and components are described in detail in the disclosure of US Pat. No. 7,670,810.

Alternative polynucleotide amplification methods useful in the present disclosure are described, for example, in Grothues et al. Nucleic Acids Res. 21(5):1321-2 (1993), Tagged PCR using a population of 2-domain primers with a constant 5'region followed by a random 3'region. A first round of amplification is performed to allow a multiplicity of initiation for heat denatured DNA based on individual hybridizations from randomly synthesized 3'regions. Due to the nature of the 3'region, the start site appears to be random throughout the genome. The unbound primer can then be removed and the primer complementary to the constant 5'region can be used for further replication.

In some embodiments, isothermal amplification can be performed using kinetic exclusion amplification (KEA), also called exclusion amplification (ExAmp). Nucleic acid libraries of the present disclosure include reacting amplification reagents to produce a plurality of amplification sites each containing a substantially clonal population of amplicons from individual target nucleic acid seeded sites. Can be made using the method. In some embodiments, the amplification reaction proceeds until a sufficient number of amplicons are produced to fill the volume of each amplification site. In this way, filling an already seeded site to volume prevents the target nucleic acid from landing and amplifying at that site, thereby producing a clonal population of amplicons at that site. In some embodiments, apparent clonality can be achieved even if the amplification site is not filled to capacity before the second target nucleic acid reaches the site. Under some conditions, amplification of the first target nucleic acid effectively produces or creates a sufficient number of copies to overwhelm the production of copies from the second target nucleic acid that is transported to the site. You can proceed to the point where it is done. For example, in an embodiment that uses a cross-linking amplification process on circular features with diameters less than 500 nm, after 14 cycles of exponential amplification for the first target nucleic acid, contamination from the second target nucleic acid at the same site is It was determined to produce an insufficient number of contaminating amplicons to adversely affect sequencing by synthetic analysis on the Illumina sequencing platform.

In some embodiments, the amplification sites in the array can, but need not, be fully clonal. Rather, for some applications, individual amplification sites can be predominantly clustered with amplicons from the first fragment-adaptor molecule and also have low levels of contaminating amplicons from the second target nucleic acid. You can An array can have one or more amplification sites with low levels of contaminating amplicons, as long as the level of contamination does not unacceptably affect subsequent use of the array. For example, if the array is used in a detection application, acceptable levels of contamination are levels that do not affect the signal-to-noise or resolution of the detection technique in an unacceptable manner. Thus, apparent clonality is generally associated with a particular use or application of arrays produced by the methods described herein. Exemplary levels of contamination that may be tolerated at individual amplification sites for a particular application include at most 0.1%, 0.5%, 1%, 5%, 10% or 25% of the contamination amplicon. But is not limited to. The array can include one or more amplification sites with these exemplary levels of contaminating amplicons. For example, 5%, 10%, 25%, 50%, 75%, or even 100% of the amplification sites in the array may have some contaminating amplicons. At least 50%, 75%, 80%, 85%, 90%, 95% or 99% or more of the sites in the array of sites or other collections may be clonal, or may be apparently clonal Is understood.

In some embodiments, kinetic exclusion can occur when the process occurs at a rate rapid enough to effectively eliminate another event or process from occurring. For example, consider the creation of a nucleic acid array in which the sites of the array are randomly seeded with fragment-adaptor molecules from solution and copies of the fragment-adaptor molecules are generated in the amplification process to fill each of the seeded sites to capacity. According to the kinetic exclusion method of the present disclosure, the seeding and amplification process can proceed simultaneously under conditions where the amplification rate exceeds the seeding rate. In this way, the relatively rapid rate at which copies are made at the site seeded with the first target nucleic acid effectively excludes the second nucleic acid from seeding the site for amplification. The kinetic exclusion amplification method can be performed as described in detail in the disclosure of US Patent Application Publication No. 2013/0338042.

Kinetic exclusion is a relatively slow rate for initiating amplification (eg, a slow rate to make a first copy of a fragment-adaptor molecule) versus a fragment-adaptor molecule (or a first of fragment-adaptor molecule). A relatively high speed for making subsequent copies of (copy) may be utilized. In the example of the previous paragraph, kinetic exclusion results in a relatively slow rate of fragment-adapter molecule seeding (eg, relatively slow diffusion or transport), versus amplification to fill the site with a copy of the fragment-adaptor seed. It occurs due to the relatively high speed. In another exemplary embodiment, kinetic exclusion is delayed in the formation of the first copy of the fragment-adapter molecule seeded at the site (eg, delayed or slow activation), as opposed to subsequent copies. Can occur due to the relatively rapid rate at which the site is filled. In this example, several different fragment-adapter molecules may be seeded at individual sites (eg, several fragment-adapter molecules may be present at each site prior to amplification). However, the first copy formation for any given fragment-adapter molecule is randomly activated such that the average rate of first copy formation is relatively slow compared to the rate at which subsequent copies are produced. Can be done. In this case, it is possible that several individual fragment-adapter molecules were seeded at individual sites, but kinetics exclusion would only allow amplification of one of these fragment-adaptor molecules. More specifically, once the first fragment-adapter molecule has been activated for amplification, the site rapidly fills its volume with that copy, whereby a copy of the second fragment-adaptor molecule is obtained. It will prevent it being made at the site.

Amplification reagents can include additional components that promote and optionally increase the rate of amplicon formation. An example is recombinase. Recombinases may facilitate amplicons formation by allowing repeated invasion/elongation. More specifically, the recombinase can facilitate entry of the fragment-adapter molecule by the polymerase and extension of the primer by the polymerase using the fragment-adaptor molecule as a template for amplicon formation. This process can be repeated as a chain reaction in which the amplicon produced from each round of invasion/extension serves as a template in the next round. This process can be performed faster than standard PCR, as no denaturation cycle (eg, via heating or chemical denaturation) is required. As such, recombinase-promoted amplification can be performed isothermally. It is generally desirable to include ATP, or other nucleotides (or, in some cases, non-hydrolyzable analogs thereof) in the recombinase-enhanced amplification reagents to facilitate amplification. Mixtures of recombinase and single chain binding (SSB) proteins are particularly useful as SSB can further facilitate amplification. Exemplary formulations for recombinase-stimulated amplification include those sold commercially by TwistDx (Cambridge, UK) as the TwistAmp kit. Useful components of the recombinase-enhanced amplification reagents and reaction conditions are described in US Pat. No. 5,223,414 and US Pat. No. 7,399,590.

Another example of a component that can be included in an amplification reagent to promote amplicon formation and, in some cases, to increase the rate of amplicon formation is helicase. Helicases can facilitate amplicon formation by allowing the chain reaction of amplicon formation. This process can be performed faster than standard PCR, as no denaturation cycle (eg, via heating or chemical denaturation) is required. As such, helicase-promoted amplification can be performed isothermally. Mixtures of helicase and single chain binding (SSB) proteins are particularly useful as SSB can further enhance amplification. Exemplary formulations for helicase-enhanced amplification include those commercially sold as BioAmp kits from Biohelix (Beverly, MA). Further, examples of useful formulations containing helicase proteins are described in US Pat. No. 7,399,590 and US Pat. No. 7,829,284, which are incorporated herein by reference in their entirety. Has been done.

Yet another example of a component that can be included in an amplification reagent to promote amplicon formation and, in some cases, increase the rate of amplicon formation is a source binding protein.

Following attachment of the fragment-adapter molecule to the surface, the sequence of the immobilized and amplified fragment-adaptor molecule is determined. Sequencing can be performed using any suitable sequencing technique, methods for determining the sequence of immobilized and amplified fragment-adapter molecules including strand resynthesis are described in the art. Are known in, for example, Bignell et al. (US Pat. No. 8,053,192), Gunderson et al. (International Publication No. 2016/130704 pamphlet), Shen et al. (U.S. Pat. No. 8,895,249), and Pipenburg et al. (US Pat. No. 9,309,502).

The methods described herein can be used in conjunction with various nucleic acid sequencing techniques. A particularly applicable technique is that in which nucleic acids are attached to fixed locations in the array such that their relative positions do not change and the array is repeatedly imaged. The embodiment in which the image is obtained in different color channels, for example, where the images are matched with different labels used to distinguish one nucleotide base type from another is particularly applicable. In some embodiments, the process for determining the nucleotide sequence of the fragment-adapter molecule can be an automated process. Preferred embodiments include synthetic sequencing ("SBS") techniques.

SBS technology generally involves the enzymatic extension of a nascent nucleic acid strand through the repeated addition of nucleotides to a template strand. In the traditional method of SBS, a single nucleotide monomer can be provided to a target nucleotide in the presence of a polymerase at each feed. However, the methods described herein may provide more than one nucleotide monomer to the target nucleic acid in the presence of a polymerase in one delivery.

In one embodiment, the nucleotide monomer comprises locked nucleic acid (LNA) or bridging nucleic acid (BNA). If the fragment-adapter molecule is produced using one or more cytosine-depleted nucleotide sequences, such as occurs when there are cytosine-depleted nucleotide sequences in the transport chain from the transposome complex, the The melting temperature of the hybridizing nucleotide monomer changes. The use of LNA or BNA in the nucleotide monomer increases the hybridization strength between the nucleotide monomer present on the immobilized fragment-adaptor molecule and the sequencing primer sequence.

SBS can use nucleotide monomers having a terminator moiety or nucleotide monomers lacking a terminator moiety. Methods utilizing nucleotide monomers lacking terminators include, for example, pyrosequencing and sequencing using γ-phosphate labeled nucleotides, as described in further detail below. In methods using nucleotide monomers lacking terminators, the number of nucleotides added in each cycle is generally variable and depends on the template sequence and the mode of nucleotide delivery. For SBS technology that uses a nucleotide monomer with a terminator moiety, the terminator can be effectively irreversible under the sequencing conditions used, as in conventional Sanger sequencing using dideoxynucleotides, or The terminator may be reversible, as is the case with the sequencing method developed by Solexa (now Illumina, Inc.).

SBS technology can use nucleotide monomers with or without labeling moieties. Thus, uptake events can be detected based on characteristics of the label (eg, fluorescence of the label); characteristics of nucleotide monomers (eg, molecular weight or charge); byproducts of nucleotide incorporation (eg, release of pyrophosphate). In embodiments where two or more different nucleotides are present in the sequencing reagent, the different nucleotides may be distinguishable from each other, or alternatively, the two or more different labels may be labeled under the detection technique used. It can be indistinguishable. For example, the different nucleotides present in the sequencing reagents can have different labels, which will have the appropriate optics, as exemplified by the sequencing method developed by Solexa (now Illumina, Inc.). Can be distinguished using.

Preferred embodiments include pyrosequencing techniques. Pyrosequencing detects the release of inorganic pyrophosphate (PPi) when specific nucleotides are incorporated into the nascent chain (Ronaghi, M., Karamohamed, S., Petersson, B., Uhlen, M. and Nyren. , P (1996) "Real-time DNA sequencing using pyrophosphate release." Analytical Biochemistry 242(1), 84-9; , 3-11; Ronaghi, M., Uhlen, M. and Nyren, P. (1998), "A sequencing method based on real-time pyrophosphate." Science 281 (5375), 363; Nos. 6,258,568 and 6,274,320). In pyrosequencing, released PPi can be detected by immediate conversion to adenosine triphosphate (ATP) by ATP sulfatase, and the level of ATP produced is detected via luciferase-producing photons. Nucleic acids to be sequenced can be attached to features in the array and the array can be imaged to capture the chemiluminescent signal generated due to the incorporation of nucleotides in the features of the array. Images can be obtained after treating the array with a particular nucleotide type (eg, A, T, C, or G). The images obtained after addition of each nucleotide type will differ as to which features in the array will be detected. These differences in the image reflect the different array content of the features on the array. However, the relative position of each feature remains unchanged in the image. The images can be stored, processed and analyzed using the methods described herein. For example, the images obtained after processing the array with each different nucleotide type are in the same manner as exemplified herein for images obtained from different detection channels for reversible terminator-based sequencing methods. It can be handled.

In another exemplary form of SBS, cycle sequencing is described, for example, in WO 04/018497 and US Pat. No. 7,057,026, which are incorporated herein by reference in their entirety. This is accomplished, for example, by the stepwise addition of reversible terminator nucleotides containing cleavable or photobleachable dye labels, as described. This approach has been commercialized by Solexa (now Illumina Inc.) and is also described in WO 91/06678 and WO 07/123,744. The availability of fluorescently labeled terminators that can reverse termination and cleave fluorescent labels facilitates efficient cyclic reversible termination (CRT) sequencing. Polymerases can also be co-engineered to efficiently incorporate and extend from these modified nucleotides.

Preferably, in the reversible terminator-based sequencing embodiment, the label does not substantially inhibit extension under SBS reaction conditions. However, the detection label may be removable, for example by cleavage or degradation. The image can be captured after incorporation of the label into the arrayed nucleic acid features. In certain embodiments, each cycle comprises co-delivery of four different nucleotide types to the array, each nucleotide type having a spectrally distinct label. Then, four images can be acquired, each using a detection channel selective for one of four different labels. Alternatively, the different nucleotide types can be added sequentially and an image of the array can be obtained during each addition step. In such an embodiment, each image will show nucleic acid features that incorporate a particular type of nucleotide. Different features may or may not be present in different images due to the different array content of each feature. However, the relative positions of the features will remain unchanged in the image. Images obtained from such a reversible terminator-SBS method can be stored, processed and analyzed as described herein. Following the image capture step, the label can be removed and the reversible terminator moiety can be removed for subsequent cycles of nucleotide addition and detection. Removing the label after it has been detected in a particular cycle and before subsequent cycles can provide the advantage of reducing background signal and crosstalk between cycles. Examples of useful labeling and removal methods are described below.

In certain embodiments, some or all of the nucleotide monomers can include reversible terminators. In such embodiments, the reversible terminator/cleavable fluorophore may comprise a fluorophore attached to the ribose moiety via a 3'ester bond (Metzker, Genome Res. 15:1767-1776 (2005)). .. Another approach separates the terminator chemistry from the cleavage of fluorescent labels (Ruparel et al., Proc Natl Acad Sci USA 102:5932-7 (2005)). Ruparel et al. Described the development of a reversible terminator using a small 3'allyl group to block extension, but could be easily deblocked by a brief treatment with a palladium catalyst. The fluorophore was attached to the base via a photocleavable linker that can be easily cleaved by exposure to long wavelength UV light for 30 seconds. Thus, either disulfide reduction or photocleavage can be used as a cleavable linker. Another approach to reversible termination is the use of spontaneous termination that occurs after placing the bulky dye on dNTPs. The presence of charged bulky dyes on dNTPs can act as effective terminators through steric and/or electrostatic hindrance. The presence of one uptake event prevents further uptake unless the dye is removed. Cleavage of the dye removes the fluorophore and effectively reverses termination. Examples of modified nucleotides are also described in US Pat. Nos. 7,427,673 and 7,057,026, which are incorporated herein by reference in their entirety.

Additional exemplary SBS systems and methods that can be used with the methods and systems described herein are disclosed in US Patent Application Publication Nos. 2007/0166705, 2006/0188901, and No. 2006/0240439, No. 2006/0281109, No. 2012/0270305, and No. 2013/0260372, US Pat. No. 7,057,026, PCT International Publication No. 05/065814, U.S. Patent Application Publication No. 2005/0100900, and PCT International Publication No. 06/064199 and International Publication No. 07/010,251.

Some embodiments may use the detection of 4 different nucleotides using less than 4 different labels. For example, SBS can be implemented utilizing the methods and systems described in incorporated materials of US 2013/0079232. As a first example, a nucleotide-type pair is detected at the same wavelength, but based on the difference in intensity for one member of the pair compared to the other, or with the signal detected for the other member of the pair. Distinctions can be made based on changes to one member of the pair that result in the appearance or disappearance of the apparent signal (eg, via chemical, photochemical, or physical modification). As a second example, three of the four different nucleotide types can be detected under certain conditions, while the fourth nucleotide type is detectable under those conditions or their Lack of label that is minimally detected under conditions (eg, minimal detection by background fluorescence). The incorporation of the first three nucleotide types into the nucleic acid can be determined based on the presence of their respective signals, and the incorporation of the fourth nucleotide type into the nucleic acid results in the absence or minimal detection of any signal. Can be determined based on As a third example, one nucleotide type may include a label that is detected in two different channels, while other nucleotide types are detected in one or less of the channels. The above three exemplary configurations are not mutually exclusive and can be used in various combinations. An exemplary embodiment combining all three examples is a first nucleotide type detected in a first channel (eg, a label detected in a first channel when excited by a first excitation wavelength). With a second nucleotide type detected in a second channel (eg, dCTP with a label detected in a second channel when excited by a second excitation wavelength), a first and a second A third nucleotide type detected in both of the two channels (eg, dTTP with at least one label detected in both channels when excited by the first and/or second excitation wavelengths), and A fluorescence-based SBS method using a fourth nucleotide type that lacks a label that is not detected or minimally detected in either channel (eg, dGTP without a label).

In addition, sequencing data can be obtained using a single channel, as described in incorporated material of US 2013/0079232. In such a so-called 1-dye sequencing approach, the first nucleotide type is labeled, but the label is removed after the first image is generated, and the second nucleotide type is labeled with the first image. Only after being labeled. The third nucleotide type retains its label in both the first and second images, and the fourth nucleotide type remains unlabeled in both images.

Some embodiments may use sequencing by ligation techniques. Such techniques use DNA ligase to incorporate oligonucleotides and identify the incorporation of such oligonucleotides. Oligonucleotides typically have different labels that correlate with the identity of particular nucleotides in the sequence to which the oligonucleotide hybridizes. As with other SBS methods, images can be obtained after treating the array of nucleic acid features with labeled sequencing reagents. Each image will show nucleic acid features that incorporate a particular type of label. Different features may or may not be present in different images due to the different array content of each feature, but the relative position of the features will remain unchanged in the image. Images obtained from the ligation-based sequencing method can be stored, processed and analyzed as described herein. Exemplary SBS systems and methods that can be used with the methods and systems described herein are US Pat. Nos. 6,969,488, 6,172,218, and No. 6,306,597.

Some embodiments are described in Nanopore Sequencing (Deamer, DW & Akeson, M. "Nanopores and nucleic acids: Prospects foruLtrapid sequencing." Trends Biotechnol. 18, 147- 151 2000; D. Branton, "Characterization of nucleic acids by nanopore analysis." Acc. Chem. Res. 35:817-825 (2002); Li, J., M. Gershow, D. Stein, E. Brand. Golovchenko, "DNA molecules and configurations in a solid-state nanopore microscope," Nat. Mater. 2:611-615 (2003)). In such an embodiment, the fragment-adapter molecule passes through the nanopore. Nanopores can be biological pore proteins such as synthetic pores or α-hemolysin. As the fragment-adaptor molecule passes through the nanopore, each base pair can be identified by measuring the variation in the electrical conductance of the pore (US Pat. No. 7,001,792; Soni, GV. & Meller, "A. Progress towerdutrafast DNA sequencing using solid-state nanopores." Clin. Chem. 53, 1996-2001 (2007), 4; -481 (2007); Cockroft, SL, Chu, J., Amorin, M. & Ghadiri, MR, "A single-molecule nanodefects depletion activity-injection. Chem. Soc. 130, 818-820 (2008), the entire disclosures of which are incorporated herein by reference). The data obtained from nanopore sequencing can be stored, processed and analyzed as described herein. In particular, the data can be treated as an image according to the exemplary processing of optical images and other images described herein.

Some embodiments may use methods that include real-time monitoring of DNA polymerase activity. Incorporation of nucleotides is described, for example, in fluorophores as described in US Pat. Nos. 7,329,492 and 7,211,414, both of which are incorporated herein by reference. Can be detected by a fluorescence resonance energy transfer (FRET) interaction between a polymerase having a γ-phosphate labeled nucleotide or nucleotide incorporation is described, for example, in US Pat. No. 7,315,019. Using zero mode waveguides as described, fluorescent nucleotide analogs and manipulations such as those described in, for example, US Pat. No. 7,405,281 and US Pat. App. Pub. No. 2008/0108082. Detected polymerase can be used. Irradiation can be limited to the zeptoliter scale volume around the surface tethered polymerase so that incorporation of fluorescently labeled nucleotides can be observed in the low background (Levene, MJ et al., “Zero- mode waveguides for single-molecule analysis at high concentrations.", Science 299, 682-686 (2003); 1026-1028 (2008); Korlach, J. et al. (2008)). The images obtained from such methods can be stored, processed and analyzed as described herein.

Some SBS embodiments include detection of protons released upon incorporation of nucleotides into extension products. For example, sequencing based on the detection of released protons may be performed using electrical detectors and related techniques commercially available from Ion Torrent (Guilford, CT, a subsidiary of Life Technologies) or US Patent Application Publication No. 2009/0026082. The sequencing methods and systems described in 2009/0127589, 2010/0137143 and 2010/0282617 can be used. The methods described herein for amplifying target nucleic acids using kinetic exclusion can be readily applied to the substrate used to detect protons. More specifically, the methods described herein can be used to produce a clonal population of amplicons used to detect protons.

The SBS method described above may advantageously be performed in a multiplex format such that a plurality of different fragment-adapter molecules are manipulated simultaneously. In particular embodiments, different fragment-adapter molecules can be treated in a common reaction vessel or on the surface of a particular substrate. This allows for convenient delivery of sequencing reagents, removal of unreacted reagents, and detection of uptake events in multiplex fashion. In embodiments that use surface-bound target nucleic acids, the fragment-adapter molecules can be in array format. In array format, the fragment-adapter molecules can typically be bound to the surface in a spatially distinct manner. Fragment-adapter molecules can be attached by direct covalent attachment, attachment to beads or other particles, or attachment to surface-attached polymerases or other molecules. The array can include a single copy of the fragment-adapter molecule at each site (also called a feature), or multiple copies with the same sequence can be present at each site or feature. Multiple copies can be produced by an amplification method, such as cross-linking amplification or emulsion PCR, as described in further detail below.

The methods described herein are, for example, at least about 10 features/cm ² , 100 features/cm ² , 500 features/cm ² , 1,000 features/cm ² , 5,000 features/cm ² , 10,. Any of a variety of densities including 000 features/cm ² , 50,000 features/cm ² , 100,000 features/cm ² , 1,000,000 features/cm ² , 5,000,000 features/cm ² or more. An array with features at can be used.

An advantage of the method described herein is that it provides rapid and efficient parallel detection of multiple cm ² . Accordingly, the present disclosure provides an integrated system capable of preparing and detecting nucleic acids using techniques known in the art such as those exemplified above. Thus, the integrated system of the present disclosure can include fluidic components capable of delivering amplification reagents and/or sequencing reagents to one or more immobilized DNA fragments, which systems include pumps, valves, reservoirs. , Fluid lines and other components. The flow cell can be configured and/or used in an integrated system for the detection of target nucleic acids. Exemplary flow cells are described, for example, in U.S. Patent Application Publication No. 2010/0111768 and U.S. Patent Application No. 13/273,666. As illustrated for the flow cell, one or more of the fluid components of the integrated system can be used for amplification and detection methods. Taking the nucleic acid sequencing embodiment as an example, one or more fluidic components of the integrated system may be sequenced for the amplification methods described herein and in the sequencing methods as exemplified above. It can be used for delivery of determination reagents. Alternatively, the integrated system can include a separate fluid system for performing the amplification method and the detection method. Examples of integrated sequencing systems capable of producing amplified nucleic acids and sequencing the nucleic acids include the MiSeq™ platform (Illumina, Inc., San Diego, Calif.) and herein by reference. It includes, but is not limited to, the devices described in incorporated US patent application Ser. No. 13/273,666.

Various compositions may occur during the performance of the methods described herein. For example, a dual-index fragment-adapter molecule comprising a dual-index fragment-adaptor molecule and a composition comprising a dual-index fragment-adapter molecule having the configuration shown in Figure 2 block vii or Figure 4 may result. A sequencing library of dual-index fragment-adaptor molecules comprising a dual-index fragment-adaptor molecule having the configuration shown in FIG. 2 block vii or FIG. 4 and a composition comprising a sequencing library can result. Such a sequencing library can be bound to an array.

The invention is illustrated by the following examples. It should be understood that the particular examples, materials, amounts, and procedures should be construed broadly in accordance with the scope and spirit of the inventions described herein.

Reagents used in Examples-Phosphate buffered saline (PBS, Thermo Fisher, Cat. 10010023)
-0.25% trypsin (Thermo Fisher, Cat. 15050057)
・Tris (Fisher, Cat. T1503)
-HCl (Fisher, Cat. A144)
-NaCl (Fisher, Cat. M-11624)
-MgCl2 (Sigma, Cat. M8226)
-Igepal (registered trademark) CA-630 (Sigma, I8896)
-Protease inhibitor (Roche, Cat. 11873580001)
PCR-clean ddH2O
-Lithium 3,5-diiodosalicylic acid (Sigma, Cat.D3635)-LAND method only-Formaldehyde (Sigma, Cat.F8775)-xSDS method-Glycine (Sigma, Cat.G8898)-xSDS method-NE buffer solution 2.1 (NEB, Cat.B7202)-xSDS method only-SDS (Sigma, Cat.L3771)-xSDS method only-Triton(TM) X-100 (Sigma, Cat.9002-93-1)-xSDS method only・DAPI (Thermo Fisher, Cat. D1306)
TD buffer from Nextera® kit (Illumina, Cat. FC-121-1031)
96 indexed cytosine-depleted transposomes (assembled using published methods, sequences shown in Table 1)
• 9-nucleotide random primer (Table 2)
・10 mM dNTP mixture (NEB, Cat. N0447)
-Klenow (3'->5' exo-) polymerase (Enzymatics, Cat. P7010-LC-L)
・200 proof ethanol ・Index added i5 and i7 PCR primers (Table 3)
-Kapa HiFi (trademark) HotStart ReadyMix
SYBR (registered trademark) Green (FMC BioProducts, Cat. 50513)
-QIAquick (registered trademark) PCR purification kit (Qiagen, Cat. 28104)
-DsDNA high sensitivity Qubit (registered trademark) (Thermo Fisher, Cat. Q32851)
・High-sensitivity bioanalyzer kit (Agilent, Cat.5067-4626)
-NextSeq sequencing kit (150-cycle high or medium)
-Unmethylated lambda DNA (Promega, Cat. D1521)
-HiSeq (registered trademark) 2500 sequencing kit (Illumina)
-HiSeq (registered trademark) X sequencing kit (Illumina)
EZ-96 DNA methylation MagPrep kit (Zymo Research, Cat D5040)
-Custom LNA sequencing primers (Table 4)
・Polyethylene glycol (PEG)
・SPRI beads

Device used in Examples 35 μM cell strainer (BD Biosciences, Cat. 352235)
• 96-well plate compatible magnetic rack • Sony SH800 cell sorter (Sony Biotechnology, Cat. SH800) or other FACS instrument capable of DAPI-based single nuclear sorting. • CFX Connect RT thermal cycler (Bio-Rad, Cat. 1855200) or other real-time thermocycler, thermomixer, Qubit® 2.0 fluorometer (Thermo Fisher, Cat. Q32866).
2100 bioanalyzer (Agilent, Cat. G2939A)
-NextSeq (registered trademark) 500 (Illumina, Cat. SY-415-1001-1)
・HiSeq (registered trademark) 2500 (Illumina)
・HiSeq (registered trademark) X (Illumina)

Oligonucleotides used in the examples

Example 1
Unmethylated lambda DNA Preparation 100 nanograms unmethylated control lambda DNA, 2X TD buffer 5 uL, NIB buffer 5uL (10mM Tris-HCl pH7.4,10MM NaCl , 3mM MgCl 2, 0.1% Igepal (Registered trademark), 1× protease inhibitor), and 4 μL of 500 nM uniquely indexed cytosine-depleted transposome were combined. The mixture was incubated at 55° C. for 20 minutes, then purified using a QIAquick® PCR purification column and eluted in 30 μL EB.

The concentration of DNA was quantified using a dsDNA sensitive Qubit 2.0 fluorometer using 2 uL of the mixture. This concentration was diluted to 17.95 pg/uL. This simulates the genomic mass of approximately 5 human cells.

Example 2
Preparation of 18% PEG SPRI Bead Mix Sera-Mag beads (1 ml) were aliquoted into low binding 1.5 mL tubes and then placed on a magnetic stand until the supernatant was clear. The beads were washed with 500 uL of a 10 mM Tris-HCl (pH 8.0) solution, the solution was removed after the supernatant became clear, and this washing step was repeated 4 times in total. The beads were resuspended in the following mixture: 18% PEG8000 (mass ratio), 1M NaCl, 10 mM Tris-HCl, pH 8.0, 1 mM EDTA, 0.05% Tween-20; at least room temperature with gentle agitation. Incubate for 1 hour, then store 18% PEG SPRI beads at 4°C. The beads were used after reaching room temperature.

Example 3
Preparation of nuclei with lithium 3,5-diiodosalicylic acid (LAND) or SDS (xSDS) NAD Preparation of Nucleation and Nucleosome Depletion When cells are in suspension cell culture, the culture is gently disrupted to disrupt cell clumps and the cells are spun at 500 xg for 5 minutes at 4°C. Pelletized and washed with 500 μL ice cold PBS.

If the cells were in adherent cell cultures, the medium was aspirated, the cells were washed with 10 ml PBS at 37°C and then at 37°C sufficient 0.25% trypsin was added to coat the monolayer. After incubation at 37° C. for 5 minutes or until 90% of the cells no longer attach to the surface, 37° C. medium was added at a 1:1 ratio to quench trypsin. Cells were pelleted by spinning at 500 xg for 5 minutes at 4°C, then washed with 500 μL ice-cold PBS.

Cells from either suspension or adherent cell cultures are pelleted by spinning at 500 xg for 5 minutes, then NIB buffer (2.5 μL 1M LIS + 197.5 μL NIB buffer). Resuspended in 200 μL of 12.5 mM LIS. After incubating on ice for 5 minutes, 800 μL of NIB buffer was added. The cells were gently passed through a 35 μM cell strainer and 5 μL of DAPI (5 mg/mL) was added.

B. Nuclear Preparation and Nucleosome Depletion xSDS Method When cells were in suspension cell culture, medium was gently triturated to disrupt cell clumps. To 10 ml cells in medium was added 406 μl 37% formaldehyde and incubated for 10 minutes at room temperature with gentle shaking. 800 microliters of 2.5 M glycine was added to the cells, incubated on ice for 5 minutes, then centrifuged at 550 xg for 8 minutes at 4°C. After washing with 10 mL ice-cold PBS, cells were resuspended in 5 mL ice-cold NIB (10 mM TrisHCl pH 7.4, 10 mM NaCl, 3 mM MgCl ₂ , 0.1% Igepal®, 1× protease inhibitor). Incubated on ice for 20 minutes with gentle mixing.

If the cells were in adherent cell cultures, the medium was aspirated, the cells were washed with 10 ml PBS at 37°C and then at 37°C sufficient 0.25% trypsin was added to coat the monolayer. After incubation at 37° C. for 5 minutes or until 90% of the cells no longer attach to the surface, 37° C. medium was added at a 1:1 ratio to quench trypsin and bring the volume to 10 ml with medium. Cells were resuspended in 10 ml medium, 406 μl 37% formaldehyde was added and incubated for 10 minutes at room temperature with gentle shaking. 800 microliters of 2.5 M glycine was added to the cells and incubated on ice for 5 minutes. Cells were centrifuged at 550 xg for 8 minutes at 4°C and washed with 10 ml ice cold PBS. After resuspending the cells in 5 mL of ice cold NIB, they were incubated on ice for 20 minutes with gentle mixing.

Cells or nuclei from either suspension cell cultures or adherent cell cultures were pelleted by spinning at 500xg for 5 minutes and washed with 900 μL of 1x NE buffer 2.1. After spinning at 500 xg for 5 minutes, the pellet was resuspended in 800 μL of 1x NE buffer 2.1 with 12 μL of 20% SDS and incubated at 42°C for 30 minutes with vigorous shaking, then 200 μL of 10% Triton ( ™ X-100 was added and incubated at 42°C for 30 minutes with vigorous shaking. The cells were gently passed through a 35 μM cell strainer and 5 μL of DAPI (5 mg/mL) was added.

Example 4
Nuclear Selection and Tagmentation Tagmentation plates were prepared with 10 μL of 1×TD buffer (for one plate: 500 μL NIB buffer+500 μL TD buffer) and 2500 single nuclei were tagged. Each well of the rotation plate. In this step, the number of nuclei per well can be varied slightly as long as the number of nuclei per well is consistent across the plate. Because the transposase index is preserved, different samples can also be multiplexed into different wells of the plate. Cells were gated according to Figure 2. After spinning down the plate at 500 xg for 5 minutes, 4OuL of 500nM uniquely indexed cytosine-depleted transposome was added to each well. After sealing, the plates were incubated for 15 minutes at 55°C with gentle shaking. The plate was then placed on ice. All wells were pooled and then passed through a 35 μM cell strainer. 5 microliters of DAPI (5 mg/mL) was added.

Example 5
Second selection of nuclei A master mix was prepared for each well with 5 uL Zymo digestion reagent (2.5 uL M-digestion buffer, 2.25 uL H2O, and 0.25 uL proteinase K). Either 10 or 22 single nuclei were sorted into each well using the most stringent sorting settings. Ten single nuclei were sorted into the wells used for the unmethylated control spike-in and 22 cells were sorted into the other wells. The plate is then centrifuged at 600 xg for 5 minutes at 4°C.

Example 6
Digestion and bisulfite conversion About 35 pg (2 uL) of unmethylated control lambda DNA pretreated with C-depleted transposomes was used to spike wells with 10 single nuclei. The plate was incubated at 50° C. for 20 minutes to digest the nuclei and 32.5 uL of freshly prepared Zymo CT conversion reagent was added according to the manufacturer's protocol. Wells were mixed by trituration and plates were centrifuged at 600 xg for 2 minutes at 4°C. The plate was placed on a thermocycler for the following steps followed by: holding at 98°C for 8 minutes, 64°C for 3.5 hours, then 4°C for less than 20 hours. Zymo MagBinding beads (5 uL) were added to each well and 150 uL of M-binding buffer was added to each well. After mixing the wells by trituration, the plate was incubated for 5 minutes at room temperature. The plate was placed on a 96-well compatible magnetic rack until the supernatant was clear.

The supernatant is removed and the wells are i) removed from the magnetic rack, ii) 100 uL of 80% ethanol is added to each well and run on the bead pellet, iii) the plate is placed back on the magnetic rack, then top It was washed with fresh 80% ethanol (by volume) by removing the clear when clear.

Desulphonation was carried out by adding 50 uL of M-desulphonation buffer to each well, thoroughly resuspending the beads by trituration, incubating for 15 minutes at room temperature, placing the plate on a magnetic rack, then the supernatant. Was achieved by clearing and removing.

The supernatant was removed. Wells i) Remove plate from magnetic rack, ii) Add 100 uL 80% ethanol to each well, run on bead pellet, iii) Put plate back on magnetic rack, then remove supernatant when clear By washing with 80% fresh ethanol (by volume).

The bead pellet was dried for about 10 minutes until the pellet began to crack visually.

Elution was achieved by adding 25 uL Zymo M-elution buffer to each well, crushing the pellet for complete dissociation, and heating the plate at 55° C. for 4 minutes.

Example 7
Linear amplification Complete eluate was used per well with the following reaction mixture: 16 uL PCR-clean H2O, 5 uL 10X NE buffer 2.1, 2 uL 10 mM dNTP mix, and 2 uL 10 uM 9-nucleotide random primers. Transferred to prepared plate.

Linear amplification was performed as follows: i) DNA was made single-stranded by incubation at 95° C. for 45 seconds, then quenched on ice and kept on ice, ii) once completely cooled, 10 U Klenow (3 '->5' exo-) polymerase is added to each well and iii) the plate is incubated at 4°C for 5 minutes, then ramped to 37°C at a rate of +1°C/15 seconds and then held at 37°C for 90 minutes. To do.

Steps i to iii were repeated 3 more times for a total of 4 rounds of linear amplification. For each amplification, the following mixture was added to the reactions in each well: 1 uL 10 uM 9-nucleotide random primer, 1 uL 10 mM dNTP mix, and 1.25 uL 4X NE buffer 2. 1. 4 rounds. Linear amplification typically significantly increases read alignment rates and library complexity compared to fewer rounds.

Wells were cleaned using the prepared 18% PEG SPRI bead mixture at 1.1X (volume concentration compared to well reaction volume) as follows. The plate was incubated for 5 minutes at room temperature, placed on a magnetic rack and the supernatant removed when clear. The bead pellet was washed with 50 uL of 80% ethanol. The remaining liquid was removed and the bead pellet was dried until cracking started. DNA was eluted in 21 uL of 10 mM Tris-Cl (pH 8.5).

Example 8
Indexed PCR Reaction Complete eluate was added to the following reaction mixture per well: 2 uL of 10 uM i7 index PCR primer, 2 uL of 10 uM i5 index PCR primer, 25 uL of 2X KAPA HiFi™ HotStart ReadyMix, and 0.5 uL of 100X SYBR. Transferred to plates prepared using ® Green I. PCR amplification was performed in a real-time thermocycler with the following cycles: 95° C. for 2 minutes (94° C. for 80 seconds, 65° C. for 30 seconds, 72° C. for 30 seconds), most of the wells were measured for SYBR. The reaction was stopped when it showed an inflection of (registered trademark) Green fluorescence. For library preparation, an inflection plateau was observed between 16 and 21 PCR cycles.

Example 9
Library Cleaning and Quantification The library was washed per well using an 18% PEGS PRI bead mixture at 0.8X (volume concentration compared to well reaction volume) as follows. The plate was incubated for 5 minutes at room temperature, placed on a magnetic rack and the supernatant was removed after clearing. The bead pellet was washed with 50 uL of 80% ethanol. The remaining liquid was removed and the bead pellet was dried until cracking started. DNA was eluted in 25 uL of 10 mM Tris-Cl (pH 8.5).

The library was pooled using 5 uL of each well and 2 uL was used to quantitate the concentration of DNA using a dsDNA Sensitive Qubit® 2.0 Fluorometer according to the manufacturer's protocol. .. The Qubit® read-out was used to dilute the library to approximately 4 ng/uL and 1 uL was run on the Sensitive Bioanalyzer 2100 according to the manufacturer's protocol. The library was then quantified for the range 200 bp to 1 kbp and the pool diluted to 1 nM for Illumina sequencing.

Example 10
Sequencing The NextSeq® 500 was set up for operation according to the manufacturer's instructions for 1 nM samples with the following modifications. The library pool was loaded at a concentration of 0.9 pM and a total volume of 1.5 mL and placed in cartridge position 10; 9 μL of 100 μM stock sequencing primer 1 was diluted in cartridge position 7 in a total of 1.5 mL of HT1 buffer. Custom primers by setting 18 μL of each custom index sequencing primer at 100 μM stock concentration into cartridge position 9 in a total of 3 mL HT1 buffer; operating NextSeq® 500 in stand-alone mode; SCIseq custom Chemistry recipe (Amini et al., 2014, Nat. Genet. 46, 1343-1349); double index selected; enter appropriate number of read cycles (150 recommended); 10 for index 1 Entered 20 cycles for Cycles and Index 2; selected custom checkboxes for all reads and indexes.

All patents, patent applications, and publications cited herein, and electronically available materials (eg, nucleotide sequence submissions in GenBank and RefSeq, and amino acid sequence submissions in SwissProt, PIR, PRF, PDB, for example). , And the translation from the annotated coding regions in GenBank and RefSeq) are incorporated herein by reference in their entirety. Supplementary materials referred to in the publication, such as supplementary tables, supplementary figures, supplementary materials and methods, and/or supplementary experimental data, are also incorporated by reference in their entirety. In the event of any inconsistency between the disclosure of the present application and the disclosure of any document incorporated herein by reference, the disclosure of the present application shall control. The foregoing detailed description and examples are provided only for clarity of understanding. Unnecessary limitations should not be understood from them. The invention is not limited to the exact details shown and described, for variations obvious to one skilled in the art are included within the invention defined by the claims.

Unless otherwise indicated, all numbers used in this specification and claims to describe the amounts of components, molecular weights, etc., are to be understood as being modified by the term "about" in all cases. is there. Therefore, unless otherwise specified, the numerical parameters described in the present specification and claims are approximate values that can be changed according to desired characteristics to be obtained by the present invention. At a minimum, and not as an attempt to limit the doctrine of equivalents to the claims, each numerical parameter is construed, at least in light of the number of significant figures reported, by applying conventional rounding techniques. Should be.

Although the numerical ranges and parameters describing the broad scope of this invention are approximations, the numerical values given in a particular example are reported as accurately as possible. However, all numerical values inherently include the ranges necessarily resulting from the standard deviation found in each test measurement.

All headings are for the convenience of the reader and should not be used to limit the meaning of the text that follows the heading, unless so specified.

Sequence listing
This application includes Sequence Listings that have been submitted electronically in ASCII format and are hereby incorporated by reference in their entireties. The ASCII copy was made on August 15, 2018, and is available as IP-1584-PCT_SL. It is named txt and has a size of 35954 kilobytes.

CROSS REFERENCE TO RELATED APPLICATIONS This application claims the benefit of US Provisional Application No. 62/516,324, filed June 7, 2017, which is hereby incorporated by reference.

Embodiments of the disclosure relate to nucleic acid sequencing. In particular, embodiments of the methods and compositions provided herein relate to generating a single cell bisulfite sequencing library and obtaining sequence data therefrom.

High cell number single cell sequencing has demonstrated its effectiveness in segregating populations within complex tissues via transcriptome, chromatin accessibility, and mutation differences. Furthermore, single cell division allowed us to assess cell differentiation trajectories in a genome-specific pattern such as DNA methylation. DNA methylation is a covalent addition to cytosine and is a cell-type specific target for activity modification in tissue expression. DNA methylation can be probed by base pair resolution using deamination chemistry of sodium bisulfite treatment.

Recent studies have optimized bisulfite sequencing as long as single cell input is required, either in single cell reduced expression bisulfite sequencing (scRRBS) or single cell whole genome bisulfite sequencing (scWGBS). Turned into However, these methods lack scalability, relying on single cell deconvolution through parallel and separate library generation, where single cell reactions are performed separately. A whole new set of reagents is required for each cell sequencing, resulting in a linear scaling of the cost for each additional cell. Due to the challenge of bisulfite conversion of DNA, droplet- or chip-based microfluidic systems have not been developed for single-cell bisulfite sequencing, and theoretically using alternative platforms. There is no viable strategy.

Provided herein are compositions and expandable high cell number single cell methylome profiling assays. Single Cell Whole Genome Sequencing (scWGBS) is improved by the single cell combinatorial indexing strategy provided herein such that cells can be processed in bulk and single cell outputs can be demultiplexed in silico. To be done. In some embodiments, the methods provided herein utilize the incorporation of transposase-based adapters, which results in increased efficacy and much higher alignment rates than exiting the method. The use of transposase to add one of the two sequencing adapters allows for much more efficient library construction with less noise reading, thus using the single cell single well method 10. Alignment rates of approximately 60% (similar to the bulk cell strategy) are produced when compared to -30%. This results in more useful sequence reads and a dramatic cost savings for the sequencing portion of the assay. The use of a single cell combinatorial indexing strategy to generate a single cell bisulfite sequencing library is demonstrated on a mixture of human and mouse cells with minimal collision rates. Also, successful deconvolution of a mixture of three human cell types has been demonstrated to achieve cell type assignment using publicly available data.

Definitions As used herein, the terms "organism", "subject" are used interchangeably and refer to animals and plants. Examples of animals are mammals such as humans.

As used herein, the term "cell type" is intended to identify a cell based on morphology, phenotype, developmental origin, or other known or discernible distinctive cellular characteristic. .. A variety of different cell types can be obtained from a single organism (or organism of the same species). Exemplary cell types include bladder, pancreatic epithelium, pancreatic α, pancreatic β, pancreatic endothelium, bone marrow lymphoblasts, bone marrow B lymphoblastoma, bone marrow macrophages, bone marrow erythroblasts, bone marrow dendritic cells, bone marrow adipocytes, bone marrow. Bone cells, bone marrow chondrocytes, promyeloblast, myelomegaloblasts, bladder, brain B lymphocytes, glial cells, neurons, brain astrocytes, neuroectodermal, brain macrophages, brain microglia, brain Epithelium, cortical neuron, brain fibroblast, breast epithelium, colon epithelium, colon B lymphocyte, breast epithelium, breast myoepithelium, breast fibroblast, colon enterocyte, cervical epithelium, ovarian epithelium, ovarian fibroblast, Ductal epithelium, tongue epithelium, tonsil dendritic, tonsil B lymphocyte, peripheral blood lymphoblast, peripheral blood T lymphoblast, peripheral blood cutaneous T lymphocyte, peripheral blood natural killer, peripheral blood B lymphoblast, Peripheral blood monocytes, peripheral blood myeloblasts, peripheral blood monoblasts, peripheral blood promyelocytes, peripheral blood macrophages, peripheral blood basophils, liver endothelium, liver obesity, liver epithelium, liver B lymphocytes, spleen endothelium, Splenic epithelium, splenic B lymphocyte, hepatocyte, liver alexander, liver fibroblast, lung epithelium, bronchial epithelium, lung fibroblast, lung B lymphocyte, lung Schwann, lung squamous epithelium, lung macrophage, lung osteoblast, Neuroendocrine, alveoli, gastric epithelium, and gastric fibroblasts include, but are not limited to.

As used herein, the term "tissue" is intended to mean the collection or aggregation of cells that work together to carry out one or more specific functions in an organism. The cells may optionally be morphologically similar. Exemplary tissues include eyes, muscles, skin, tendons, veins, arteries, blood, heart, spleen, lymph nodes, bone, bone marrow, lungs, bronchi, trachea, intestine, small intestine, large intestine, colon, rectum, salivary gland, Tongue, gallbladder, appendix, liver, pancreas, brain, stomach, skin, kidney, ureter, bladder, urethra, gonad, testis, ovary, uterus, fallopian tube, thymus, pituitary gland, thyroid, adrenal gland, or parathyroid gland However, the present invention is not limited to these. The tissue can be from any of various organs of humans or other organisms. The tissue can be healthy tissue or unhealthy tissue. Examples of unhealthy tissues include various malignancies with abnormal methylation, such as lung, breast, colorectal, prostate, nasopharyngeal, stomach, testis, skin, nervous system, bone, ovary, liver, blood tissue. , But not limited to malignant tumors in the pancreas, uterus, kidneys, lymphoid tissues and the like. Malignant tumors can be of various histological subtypes, eg, carcinoma, adenocarcinoma, sarcoma, fibroadenocarcinoma, neuroendocrine, or undifferentiated.

As used herein, the term "compartment" is intended to mean a range or volume that separates or isolates something from another. Exemplary compartments include, but are not limited to, vials, tubes, wells, droplets, boluses, beads, containers, surface features, or ranges or volumes separated by physical forces such as fluid flow, magnetic forces, electric currents. Not limited. In one embodiment, the compartments are the wells of a multi-well plate such as a 96-well plate or a 384-well plate.

As used herein, "transposome complex" refers to a nucleic acid that contains an integrative enzyme and an integrative recognition site. A "transposome complex" is a functional complex formed by a transposase and a transposase recognition site capable of catalyzing a rearrangement reaction (see, for example, Gunderson et al., WO 2016/130704). Examples of incorporated enzymes include, but are not limited to, integrase or transposase and the like. Examples of integrated recognition sites include, but are not limited to, transposase recognition sites.

As used herein, the term "nucleic acid" is intended to be consistent with its use in the art and includes naturally occurring nucleic acids or functional analogs thereof. Particularly useful functional analogs can hybridize to nucleic acids in a sequence specific manner, or can be used as templates for the replication of specific nucleotide sequences. Naturally occurring nucleic acids generally have a backbone that contains phosphodiester bonds. The analog structure can have alternative backbone bonds, including any of a variety known in the art. Naturally occurring nucleic acids generally have a deoxyribose sugar (eg, found in deoxyribonucleic acid (DNA)) or a ribose sugar (eg, found in ribonucleic acid (RNA)). The nucleic acid can include any of the various analogs of these sugar moieties known in the art. Nucleic acids can include natural or unnatural bases. In this regard, the natural deoxyribonucleic acid can have one or more bases selected from the group consisting of adenine, thymine, cytosine or guanine, the ribonucleic acid being selected from the group consisting of uracil, adenine, cytosine or guanine. It may have one or more bases as described. Useful non-natural bases that can be included in nucleic acids are known in the art. Examples of non-natural bases include locked nucleic acids (LNA) and bridging nucleic acids (BNA). LNA and BNA bases can be incorporated into DNA oligonucleotides to increase oligonucleotide hybridization strength and specificity. LNA and BNA bases and the use of such bases are known and routine to those of skill in the art.

As used herein, the term "target", when used with reference to a nucleic acid, is intended as the semantic identifier of the nucleic acid in the context of the methods or compositions presented herein, or otherwise. It does not necessarily limit the composition or function of the nucleic acid beyond what is explicitly indicated. The target nucleic acid can be essentially any nucleic acid of known or unknown sequence. It can be, for example, a fragment of genomic DNA or cDNA. Sequencing can result in the determination of the sequence of all or part of a target molecule. The target can be derived from a primary nucleic acid sample such as a nucleus. In one embodiment, the target can be processed into a template suitable for amplification by placing a universal sequence at the end of each target fragment. Targets can also be obtained from the primary RNA sample by reverse transcription into cDNA.

As used herein, the term "generic", when used to describe a nucleotide sequence, refers to a region of sequence common to two or more nucleic acid molecules, which molecule also has regions of sequence that differ from each other. Point to. A universal sequence that is present in different members of a collection of molecules can be used to capture a plurality of different nucleic acids using a population of universal capture nucleic acids, e. Can be enabled. Non-limiting examples of universal capture sequences include sequences that are the same or complementary to the P5 and P7 primers. Similarly, a universal sequence that is present in a different member of a collection of molecules uses a portion of the universal sequence, for example, a population of universal primers that are complementary to a universal anchor sequence, to replicate or amplify multiple different nucleic acids. May enable. Thus, the capture oligonucleotide or universal primer comprises a sequence that can specifically hybridize to the universal sequence.

The terms "P5" and "P7" may be used when referring to amplification primers, such as capture oligonucleotides. The terms "P5'" (P5' primer) and "P7'" (P7' primer) refer to the complements of P5 and P7, respectively. It will be appreciated that any suitable amplification primer can be used in the methods presented herein, and the use of P5 and P7 is an exemplary embodiment only. The use of amplification primers such as P5 and P7 on flow cells is described in WO2007/010251, WO2006/064199, WO2005/065814, WO2015/106941. It is known in the art, as exemplified by the disclosures of the pamphlet, WO 1998/044151 and WO 2000/018957. For example, any suitable forward amplification primer, whether immobilized or in solution, is useful in the methods presented herein for hybridization to complementary sequences and amplification of sequences. possible. Similarly, any suitable reverse amplification primer, whether immobilized or in solution, is useful in the methods provided herein for hybridization to complementary sequences and amplification of sequences. Can be Those skilled in the art will understand how to design and use suitable primer sequences for the capture and/or amplification of nucleic acids as presented herein.

As used herein, the term "primer" and its derivatives generally refer to any nucleic acid capable of hybridizing to a target sequence of interest. Typically, the primer functions as a substrate whose nucleotides can be polymerized by a polymerase, but in some embodiments, the primer is incorporated into a synthesized nucleic acid strand and another primer hybridizes to the synthetic nucleic acid. It may provide a site for prime synthesis of a new strand that is complementary to the molecule. Primers can include any combination of nucleotides or analogs thereof. In some embodiments, the primer is a single stranded oligonucleotide or polynucleotide. The terms "polynucleotide" and "oligonucleotide" are used interchangeably herein to refer to polymeric forms of nucleotides of any length, including ribonucleotides, deoxyribonucleotides, their analogs, or their It may include a mixture. The term includes, as equivalents, either DNA or RNA analogs made from nucleotide analogs and is applicable to single-stranded (eg, sense or antisense) and double-stranded polynucleotides. Should be understood. The term as used herein also includes cDNA, which is complementary or copy DNA produced from an RNA template, for example by the action of reverse transcriptase. This term refers only to the primary structure of the molecule. Thus, the term includes triple-stranded, double-stranded and single-stranded deoxyribonucleic acid (“DNA”), as well as triple-stranded, double-stranded and single-stranded ribonucleic acid (“RNA”).

As used herein, the term "adapter" and its derivatives (eg, universal adapter) generally refers to any linear oligonucleotide that can be linked to the nucleic acid molecules of the present disclosure. In some embodiments, the adapter is not substantially complementary to the 3'or 5'ends of any target sequence present in the sample. In some embodiments, suitable adapter lengths range from about 10-100 nucleotides, about 12-60 nucleotides and about 15-50 nucleotides in length. In general, the adapter can include any combination of nucleotides and/or nucleic acids. In some aspects, the adapter can include one or more cleavable groups at one or more positions. In another aspect, the adapter can include a sequence that is substantially identical or substantially complementary to at least a portion of a primer, eg, a universal primer. In some embodiments, the adapter can include a barcode or tag to aid downstream error correction, identification, or ordering. The terms "adaptor" and "adapter" are used interchangeably.

As used herein, the term "each", when used in reference to a collection of items, is intended to identify individual items within the collection, but the context clearly indicates otherwise. Unless it does, it does not necessarily refer to every item in the collection.

As used herein, the term "transport" refers to the movement of molecules through a fluid. The term may include passive transport, such as migration of molecules along their concentration gradient (eg, passive diffusion). The term can also include active transport, where molecules can move along or against their concentration gradient. Thus, transport can include applying energy to move one or more molecules in a desired direction or to a desired location, such as an amplification site.

As used herein, "amplify", "amplification" or "amplification reaction" and their derivatives generally mean that at least a portion of a nucleic acid molecule is replicated or copied into at least one additional nucleic acid molecule. Refers to any action or process. The additional nucleic acid molecule optionally comprises a sequence that is substantially identical or substantially complementary to at least some portion of the template nucleic acid molecule. Template nucleic acid molecules can be single-stranded or double-stranded, and additional nucleic acid molecules can be single-stranded or double-stranded independently. Amplification optionally involves linear or exponential replication of the nucleic acid molecule. In some embodiments, such amplification can be performed using isothermal conditions, and in other embodiments, such amplification can include thermocycling. In some embodiments, the amplification is a multiplex amplification that involves the simultaneous amplification of multiple target sequences in a single amplification reaction. In some embodiments, "amplifying" comprises amplifying at least some portions of DNA and RNA based nucleic acids, alone or in combination. The amplification reaction can include any of the amplification processes known to those of skill in the art. In some embodiments, the amplification reaction comprises the polymerase chain reaction (PCR).

As used herein, "amplification conditions" and derivatives thereof generally refer to conditions suitable for amplifying one or more nucleic acid sequences. Such amplification can be linear or exponential. In some embodiments, amplification conditions can include isothermal conditions, or can include thermocycling conditions, or a combination of isothermal and thermocycling conditions. In some embodiments, suitable conditions for amplifying one or more nucleic acid sequences include polymerase chain reaction (PCR) conditions. Typically, amplification conditions are for amplifying nucleic acids such as one or more target sequences or for amplifying amplified target sequences linked to one or more adapters, eg, adapter-ligated amplification target sequences. Refers to sufficient reaction mixture. Generally, amplification conditions include a catalyst for amplification or nucleic acid synthesis, such as a polymerase; a primer with some complementarity to the nucleic acid to be amplified; and nucleotides to facilitate extension of the primer after hybridizing to the nucleic acid. , For example, deoxyribonucleotide triphosphate (dNTP). Amplification conditions may require hybridization or annealing of the primer to the nucleic acid, extension of the primer, and denaturing steps in which the extended primer is separated from the nucleic acid sequence undergoing amplification. Typically, the amplification conditions can, but need not, include thermocycling, and in some embodiments, the amplification conditions include multiple cycles in which the steps of annealing, extension, and separation are repeated. Typically, the amplification state will contain cations such as Mg ²⁺ or Mn ²⁺ and may also contain various modifiers of ionic strength.

As used herein, "reamplification" and its derivatives generally mean that at least a portion of the amplified nucleic acid molecule is from any suitable amplification process (in some embodiments, "secondary"). Referred to as amplification), thereby producing a reamplified nucleic acid molecule. The secondary amplification need not be the same as the original amplification process by which the amplified nucleic acid molecule was produced, nor that the amplified nucleic acid molecule is exactly the same or is completely complementary to the amplified nucleic acid molecule. Nonetheless, it is only necessary that the reamplified nucleic acid molecule comprises at least part of the amplified nucleic acid molecule or its complement. For example, reamplification can include different amplification conditions and/or the use of different primers, including different target-specific primers than the primary amplification.

As used herein, the term “polymerase chain reaction” (“PCR”) refers to the method of Mullis US Pat. Nos. 4,683,195 and 4,683,202, These describe methods for increasing the concentration of a segment of a polynucleotide of interest in a mixture of genomic DNA without cloning or purification. This process for amplifying a polynucleotide of interest involves introducing a large excess of two oligonucleotide primers into a DNA mixture containing the desired polynucleotide of interest, followed by a series of thermal cycles in the presence of DNA polymerase. It consists of The two primers are complementary to each strand of the double-stranded polynucleotide of interest. The mixture is first denatured at higher temperature and then the primer is annealed to the complementary sequence within the polynucleotide molecule of interest. After annealing, the primers are extended with a polymerase to form new pairs of complementary strands. The steps of denaturation, primer annealing and polymerase extension can be repeated many times (called thermocycling) to obtain high concentrations of the amplified segment of the desired polynucleotide of interest. The length of the amplified segment (amplicon) of the desired polynucleotide of interest is determined by the relative position of the primers with respect to each other, and thus this length is a controllable parameter. By repeating this process, the method is referred to as the "polymerase chain reaction" (hereinafter "PCR"). Since the desired amplified segment of the polynucleotide of interest will be the predominant (in terms of concentration) nucleic acid sequence in the mixture, they are said to be "PCR amplified." In a modification to the above method, the target nucleic acid molecule can be PCR amplified using multiple different primer pairs, optionally one or more primer pairs per target nucleic acid molecule of interest, thereby providing a multiplex PCR reaction. Form.

As defined herein, "multiplex amplification" refers to the selective and non-random amplification of two or more target sequences in a sample using at least one target-specific primer. In some embodiments, multiplex amplification is performed such that some or all of the target sequences are amplified in a single reaction vessel. The “plexi” or “plex” of a given multiplex amplification generally refers to the number of different target-specific sequences amplified during that single multiplex amplification. In some embodiments, the plexi can be about 12, 24, 48, 96, 192, 384, 768, 1536, 3072, 6144 or more. Several different methodologies, such as gel electrophoresis followed by densitometry, bioanalyzer quantification, or quantitative PCR, hybridization with labeled probes; biotinylated primer incorporation, followed by avidin-enzyme conjugate detection; It is also possible to detect the amplified target sequence (by incorporation of 32 P-labeled deoxynucleotide triphosphate into the amplified target sequence).

As used herein, "amplified target sequence" and its derivatives generally refer to nucleic acid produced by an amplified target sequence using target-specific primers and the methods provided herein. Refers to an array. The amplified target sequence can be either the same sense (ie, plus strand) or antisense (ie, minus strand) with respect to the target sequence.

As used herein, the terms “link”, “link” and their derivatives generally refer to covalently linking two or more molecules together, eg, two or more. Refers to the process for covalently linking nucleic acid molecules to each other. In some embodiments, linking comprises linking a nick between adjacent nucleotides of a nucleic acid. In some embodiments, the ligation comprises forming a covalent bond between the end of the first nucleic acid molecule and the end of the second nucleic acid molecule. In some embodiments, the linking forms a covalent bond between the 5'phosphate group of one nucleic acid and the 3'hydroxyl group of a second nucleic acid, thereby forming a linked nucleic acid molecule. Can be included. In general, for the purposes of this disclosure, the amplified target sequence may be ligated to an adapter to produce an adapter ligated amplified target sequence.

As used herein, "ligase" and its derivatives generally refer to any agent capable of catalyzing the linking of two substrate molecules. In some embodiments, the ligase comprises an enzyme capable of catalyzing the nicking between adjacent nucleotides of a nucleic acid. In some embodiments, the ligase catalyzes the formation of a covalent bond between the 5'phosphate of one nucleic acid molecule and the 3'hydroxyl of another nucleic acid molecule, thereby forming a linked nucleic acid molecule. Including enzymes that can. Suitable ligases can include, but are not limited to, T4 DNA ligase, T4 RNA ligase, and E. coli DNA ligase.

As used herein, "linking conditions" and derivatives thereof generally refer to conditions suitable for linking two molecules together. In some embodiments, the ligation conditions are suitable to seal nicks or gaps between nucleic acids. The term nick or gap, as used herein, is consistent with its use in the art. Typically, the nicks or gaps can be linked in the presence of an enzyme (eg, ligase) at a suitable temperature and pH. In some embodiments, T4 DNA ligase is capable of binding nicks between nucleic acids at temperatures of about 70-72°C.

The term “flow cell” as used herein refers to a chamber containing a solid surface through which one or more fluid reagents can flow. Examples of flow cells and associated fluidic systems and detection platforms that can be readily used in the methods of the present disclosure are incorporated herein by reference, eg, Bentley et al. , Nature 456:53-59 (2008), WO 04/018497; US Pat. No. 7,057,026; WO 91/06678; WO 07/123744; US US Pat. No. 7,329,492; US Pat. No. 7,211,414; US Pat. No. 7,315,019; US Pat. No. 7,405,281; and US patents. It is described in Japanese Patent Application Publication No. 2008/01082.

As used herein, the term "amplicon," when used in reference to a nucleic acid, means a product that copies a nucleic acid, which product is the same as or complementary to at least a portion of the nucleotide sequence of the nucleic acid. Having a nucleotide sequence that is targeted. An amplicon is any of various amplification methods that use a nucleic acid or its amplicon as a template, including, for example, polymerase extension, polymerase chain reaction (PCR), rolling circle amplification (RCA), ligation extension, or ligation chain reaction. Can be produced by An amplicon can be a nucleic acid molecule that has a single copy of a particular nucleotide sequence (eg, a PCR product) or multiple copies of a nucleotide sequence (eg, a concatemer product of RCA). The first amplicon of the target nucleic acid is typically a complementary copy. Subsequent amplicons are copies made from the target nucleic acid or from the first amplicon after the production of the first amplicon. The subsequent amplicon can have a sequence that is substantially complementary to, or is substantially identical to, the target nucleic acid.

As used herein, the term "amplification site" refers to a site in or on an array where one or more amplicons can be produced. The amplification site can be further configured to include, retain, or attach to at least one amplicon produced at that site.

As used herein, the term "array" refers to a population of sites that can be distinguished from each other according to their relative position. Different molecules at different sites of the array can be distinguished from each other according to the position of the sites in the array. Individual sites in the array may contain one or more molecules of a particular type. For example, a site may contain a single target nucleic acid molecule having a particular sequence, or a site may contain several nucleic acid molecules having the same sequence (and/or its complementary sequence). The sites of the array can be different features located on the same substrate. Exemplary features include, but are not limited to, wells within the substrate, beads (or other particles) within or on the substrate, protrusions from the substrate, ridges on the substrate, or channels within the substrate. .. The sites of the array can be separate substrates, each with a different molecule. Different molecules attached to separate substrates can be identified according to the position of the substrate on the surface with which the substrate is associated, or according to the position of the substrate in a liquid or gel. Exemplary arrays in which the discrete substrates are located on the surface include, but are not limited to, those with beads in the wells.

As used herein, the term "volume", when used in reference to a site and nucleic acid material, means the maximum amount of nucleic acid material that can occupy the site. For example, the term can refer to the total number of nucleic acid molecules that can occupy a site in a particular state. Other measures can be used as well, including, for example, the total mass of nucleic acid material that can occupy a site in a particular state, or the total number of copies of a particular nucleotide sequence. Typically, the volume of the site for the target nucleic acid is substantially equivalent to the volume of the site for the amplicon of the target nucleic acid.

As used herein, the term "capture agent" refers to a material, chemical, molecule, or portion thereof that is capable of attaching, retaining or binding to a target molecule (eg, target nucleic acid). Exemplary capture agents include a capture nucleic acid that is complementary to at least a portion of the target nucleic acid (also referred to herein as a capture oligonucleotide), a receptor capable of binding to the target nucleic acid (or a binding moiety attached thereto). Can form a covalent bond with a member of a body-ligand binding pair (eg, avidin, streptavidin, biotin, lectin, carbohydrate, nucleic acid binding protein, epitope, antibody, etc.) or a target nucleic acid (or binding moiety attached thereto). These include, but are not limited to, possible chemical reagents.

As used herein, the term "clonal population" refers to a population of nucleic acids that is homogenous with respect to a particular nucleotide sequence. Homogeneous sequences are typically at least 10 nucleotides in length, but can be longer, including at least 50, 100, 250, 500 or 1000 nucleotides in length. A clonal population can be derived from a single target nucleic acid or template nucleic acid. Typically, all of the nucleic acids in a clonal population will have the same nucleotide sequence. It is understood that a small number of mutations can occur in the clonal population (eg due to amplification artifacts) without departing from clonality.

As used herein, “providing” in the context of a composition, article, nucleic acid, or nucleus refers to making a composition, article, nucleic acid, or nucleus, composition, article, nucleic acid, or nucleus. Or otherwise obtaining the compound, composition, article, article, or nucleus.

The term "and/or" means one or all of the listed elements or a combination of any two or more of the listed elements.

The words "preferred" and "preferably" refer to embodiments of the invention that may afford certain benefits, under certain circumstances. However, under the same or different circumstances, other embodiments may be preferred. Furthermore, the description of one or more preferred embodiments does not indicate that another embodiment is not useful, and is not intended to exclude another embodiment from the scope of the present invention.

The term "comprising" and variations thereof do not have a limiting meaning where these terms appear in the description and claims.

Embodiments described herein in terms such as "comprises" or "comprising" are everywhere else described by the terms "consisting of" and/or "consisting essentially of." It is understood that similar embodiments are also provided.

Unless otherwise specified, "a," "an," "the," and "at least one" are used interchangeably and mean one or more.

Further, in the present specification, the description of the numerical range by the end point includes all the numbers included in the range (for example, 1 to 5 are 1, 1.5, 2, 2.75, 3, 3). .80, 4, 5, etc.).

For any method disclosed herein, including separate steps, the steps may occur in any feasible order. Further, if desired, any combination of two or more steps may be performed simultaneously.

Throughout this specification, reference to "an embodiment," "an embodiment," "a particular embodiment," "some embodiments," or the like refers to a particular feature described in connection with that embodiment. , Composition, composition, or property is included in at least one embodiment of the present disclosure. Thus, the appearances of such phrases in various places throughout the specification are not necessarily all referring to the same embodiment of the disclosure. Furthermore, the particular features, configurations, compositions, or characteristics may be combined in any suitable manner in one or more embodiments.

The following detailed description of specific embodiments of the present disclosure can be best understood when read in conjunction with the following drawings.

FIG. 6 shows a general block diagram of a general exemplary method for single cell combination index addition according to the present disclosure. FIG. 1 shows a schematic diagram of one embodiment of a method for single cell combinatorial indexing generally shown in FIG. Figure 3 shows a schematic of an exemplary embodiment of the fragment-adaptor molecule after linear amplification. Figure 3 shows a schematic of an exemplary embodiment of a fragment-adapter molecule after addition of a universal adapter.

The schematic is not necessarily isometric. Like numbers used in the figures refer to like components, steps, and the like. It will be appreciated, however, that the use of numbers to refer to components in a given figure is not intended to limit components in other figures that are labeled with the same number. Furthermore, the use of different numbers to refer to components is not intended to indicate that different numbered components may not be the same or similar to other numbered components.

The methods provided herein include providing isolated nuclei from multiple cells (Figure 1, block 12). The cell may be derived from any organism, and may be derived from any cell type or any tissue of the organism. The method may further comprise dissociating the cells (Figure 2, block i) and/or isolating the nucleus (Figure 2, block ii). Methods for isolating nuclei from cells are well known and routine to those of skill in the art. The number of nuclei can be at least 2. The upper limit depends on the practical limits of the equipment (eg, multiwell plate) used in the other steps of the methods described herein. For example, in one embodiment, the number of nuclei is 1,000,000 or less, 100,000,000 or less, 10,000,000 or less, 1,000,000 or less, 10,000 or less, or 1, 000 or less. Those skilled in the art will recognize that the nucleic acid molecule in each nucleus represents the entire genetic complement of the organism and is a genomic DNA molecule containing both intron and exon sequences and non-coding regulatory sequences such as promoter and enhancer sequences. Will.

In one embodiment, the nucleus comprises nucleosomes bound to genomic DNA. Such nuclei may be useful in methods that do not sequence the entire genome of the cell, such as sciATAC-seq. In another embodiment, the isolated nuclei are subjected to conditions that deplete the nucleosomal nuclei to produce nucleosomal depleted nuclei (FIG. 1, block 13, and FIG. 2, block ii). Such nuclei may be useful in methods aimed at determining the total genomic DNA sequence of a cell. In one embodiment, the conditions used for nucleosome depletion maintain the integrity of the isolated nucleus. Methods for generating nucleosome-depleted nuclei are known to those of skill in the art (see, eg, Vitak et al., 2017, Nature Methods, 14(3):302-308). In one embodiment, the conditions are chemical treatments, including treatment with chaotropic agents that can disrupt nucleic acid-protein interactions. Examples of useful chaotropic agents include, but are not limited to, lithium diiodosalicylate. In another embodiment, the conditions are chemical treatments, including treatment with detergents that can disrupt nucleic acid-protein interactions. An example of a useful detergent includes, but is not limited to, sodium dodecyl sulfate (SDS). In some embodiments, when a detergent such as SDS is used, the cells from which the nuclei are isolated are treated with a crosslinker prior to isolation. Useful examples of cross-linking agents include, but are not limited to, formaldehyde.

The methods provided herein include partitioning a subset of nuclei (eg, nucleosome depleted nuclei) into a first plurality of compartments (FIG. 1, block 14 and FIG. 2, left side schematic). The number of nuclei present in the subset, and thus the number of nuclei present in each compartment, can be at least one. In one embodiment, the number of nuclei present in the subset is 2,000 or less. Methods for partitioning nuclei into subsets are well known and routine to those of skill in the art. An example includes, but is not limited to, fluorescence activated nuclear sorting (FANS).

Each compartment contains a transposome complex. The transposase complex, which is a transposase bound to a transposase recognition site, can insert the transposase recognition site into a target nucleic acid in the nucleus in a process sometimes called “tagmentation”. In some such insertion events, one strand of the transposase recognition site can be transferred to the target nucleic acid. Such chains are called "transport chains". In one embodiment, the transposome complex comprises a dimeric transposase with two subunits and two discontinuous transposon sequences. In another embodiment, the transposase comprises a dimeric transposase having two subunits and a continuous transposon sequence.

Some embodiments include an overactive Tn5 transposase and a Tn5-type transposase recognition site (Goryshin and Reznikoff, J. Biol. Chem., 273:7367 (1998)), or a Mu transposase that includes a MuA transposase and R1 and R2 terminal sequences. The use of recognition sites (Mizuchi, K., Cell, 35:785, 1983; Savirahti, H, et al., EMBO J., 14:4893, 1995) may be involved. The Tn5 mosaic end (ME) sequence can also be optimized and used by those skilled in the art.

Further examples of transposition systems that can be used in certain embodiments of the compositions and methods provided herein are Staphylococcus aureus Tn552 (Colegio et al., J. Bacteriol., 183:2384). -8, 2001; Kirby C et al., Mol. Microbiol., 43:173-86, 2002), Ty1 (Device & Boeke, Nucleic Acids Res., 22:3765-72, 1994 and International Publication No. 95/23875. No. Pamphlet), transposon Tn7 (Craig, NL, Science. 271:1512, 1996; Craig, NL, Review in: Curr Top Microbiol Immunol., 204:27-48, 1996), Tn/O and IS10 (Kleckner). N, et al., Curr Top Microbiol Immunol., 204:49-82, 1996), Mariner transposase (Lampe DJ, et al., EMBO J., 15:5470-9, 1996), Tc1 (Plasterk RH. , Curr. Topics Microbiol. Immunol., 204:125-43, 1996), P element (Gloor, GB, Methods Mol. Biol., 260:97-114, 2004), Tn3 (Ichikawa & Ohtsubo, J Bio. Chem. 265: 18829-32, 1990), bacterial insertion sequence (Ohtsubo & Sekine, Curr. Top. Microbiol. Immunol. 204:1-26, 1996), retrovirus (Brown, et al., Proc Natl Acad Sci USA). , 86:2525-9, 1989), and yeast retrotransposons (Boeke & Centers, Annu Rev Microbiol. 43:403-34, 1989). Further examples include engineered versions of IS5, Tn10, Tn903, IS911, and transposase family enzymes (Zhang et al., (2009) PLoS Genet. 5:e1000689.Epub 2009 Oct 16; Wilson C. et al( 2007) J. Microbiol. Methods 71:332-5).

Another example of an integrase that can be used in the methods and compositions provided herein is a retroviral integrase such as an integrase from HIV-1, HIV-2, SIV, PFV-1, RSV. And the integrase recognition sequences of such retroviral integrases.

Transposon sequences useful in the methods and compositions provided herein are disclosed in US Patent Application Publication No. 2012/0208705, US Patent Application Publication No. 2012/0208724, and International Patent Application Publication No. 2012/061832. It is provided in the issue brochure. In some embodiments, the transposon sequence comprises a first transposase recognition site, a second transposase recognition site, and an index that lies between these two transposase recognition sites.

Some transposome complexes useful herein include transposases having two transposon sequences. In some such embodiments, the two transposon sequences are not linked to each other, in other words the transposon sequences are discontinuous with each other. Examples of such transposomes are known in the art (see, eg, US Patent Application Publication No. 2010/0120098).

In some embodiments, the transposome complex comprises a transposon sequence nucleic acid that binds to two transposase subunits to form a "loop complex" or "loop transposome." In one example, the transposome comprises dimeric transposase and transposon sequences. The looped complex can ensure that the transposon is inserted into the target DNA without fragmenting the target DNA while maintaining the ordering information of the original target DNA. As will be appreciated, the looped structure can insert a desired nucleic acid sequence, such as an index, into the target nucleic acid while maintaining the physical connectivity of the target nucleic acid. In some embodiments, the transposon sequences of the looped transposome complex can include fragmentation sites such that the transposon sequences can be fragmented to produce a transposome complex that contains two transposon sequences. Such transposome complexes are useful to ensure that the flanking target DNA fragments into which the transposon is inserted receive a coding combination that can be unambiguously assembled in later steps of the assay.

The transposome complex also contains at least one index sequence, also called the transposase index. The index sequence exists as part of the transposon sequence. In one embodiment, the index sequence may be on a transport strand that is the strand of the transposase recognition site that is transported to the target nucleic acid. Index sequences, also called tags or barcodes, are useful as marker features in the compartment where a particular target nucleic acid was present. The index sequence of the transposome complex differs from compartment to compartment. Therefore, in this embodiment, the index is a nucleic acid sequence tag attached to each of the target nucleic acids present in a particular compartment, the presence of which indicates the compartment in which the population of nuclei was present at this stage of the method, or Or used to identify.

Index sequences are up to 20 nucleotides in length, eg 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19. , 20. The 4-nucleotide tag offers the possibility to multiplex 256 samples on the same array and the 6-base tag allows 4096 samples to be processed on the same array.

In one embodiment, the transport strand can also include a universal sequence, a first sequencing primer sequence, or a combination thereof. Universal sequences and sequencing primer sequences are described herein. Thus, in some embodiments in which the transfer strand is transferred to the target nucleic acid, the target nucleic acid comprises a transposase index and also comprises a universal sequence, a first sequencing primer sequence, or a combination thereof.

In one embodiment, the transport chain cytosine nucleotides are methylated. In another embodiment, the nucleotides of the transport strand do not include cytosine. Any sequence present on the transport chain, including such a transport chain and a transposase index sequence, a universal sequence, and/or a first sequencing primer sequence, can be referred to as cytosine depleted. The use of cytosine-depleted nucleotide sequences in the transposome complex has no significant effect on transposase efficiency.

The method also includes generating indexed kernels (FIG. 1, block 15, and FIG. 2, block iii). In one embodiment, generating the indexed nuclei comprises fragmenting nucleic acids present in a subset of nucleosome depleted nuclei (eg, nucleic acids present in each compartment) into multiple nucleic acid fragments. In one embodiment, fragmentation of nucleic acids is accomplished by using the fragmentation sites present in the nucleic acid. Typically, the fragmentation site is introduced into the target nucleic acid by using the transposome complex. For example, the looped transposome complex can include a fragmentation site. Fragmentation sites can be used to break physical rather than informative relationships between index sequences inserted into a target nucleic acid. Cleavage may be by biochemical, chemical or other means. In some embodiments, the fragmentation site can include nucleotides or nucleotide sequences that can be fragmented by various means. Examples of fragmentation sites are a restriction endonuclease site, at least one ribonucleotide cleavable by RNAse, a nucleotide analog cleavable in the presence of certain chemical agents, cleavable by treatment with periodate. Examples include, but are not limited to, diol bonds, disulfide groups that are cleavable by chemical reducing agents, cleavable moieties that can be subject to photochemical cleavage, and peptides that are cleavable by peptidase enzymes or other suitable means (eg, US See Patent Application Publication No. 2012/0208705, US Patent Application Publication No. 2012/0208724, and International Publication No. WO 2012/061832 pamphlet). The result of the fragmentation is a population of indexed nuclei, each nucleus containing a nucleic acid fragment, wherein the nucleic acid fragment comprises, in at least one strand, an index sequence that represents a particular compartment.

Indexed kernels from multiple compartments can be combined (FIG. 1, block 16, and FIG. 2, left-hand schematic). For example, indexed nuclei from 2 to 96 compartments (if 96 well plates are used) or 2 to 384 compartments (if 384 well plates are used) are combined. A subset of these combined indexed nuclei, referred to herein as pooled indexed nuclei, is then distributed to the second plurality of partitions. The number of nuclei present in the subset, and thus in each compartment, is based in part on the desire to reduce index collisions, which leads to the same transposase index ending in the same compartment at this step of the method. It is the existence of two nuclei. In this embodiment, the number of nuclei present in the subset is between 2 and 30, for example 2,3,4,5,6,7,8,9,10,11,12,13,14,15,16. , 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30. In one embodiment, the number of nuclei present in the subset is 20-24, eg 22. Methods for partitioning nuclei into subsets are well known and routine to those of skill in the art. An example includes, but is not limited to, fluorescence activated nuclear sorting (FANS).

The distributed indexed nuclei are processed to identify methylated nucleotides (FIG. 1, block 17, and FIG. 2, block iv). Methylation of sites such as CpG dinucleotide sequences can be measured using any of the various techniques used in the art for analysis of such sites. One useful method is the identification of methylated CpG dinucleotide sequences. Identification of methylated CpG dinucleotide sequences was determined using techniques based on cytosine conversion, which rely on methylation state-dependent chemical modification of CpG sequences within isolated genomic DNA or fragments thereof, followed by DNA sequence analysis. To be done. Chemical reagents capable of distinguishing between methylated and unmethylated CpG dinucleotide sequences include hydrazine, which cleaves nucleic acids, and bisulfite. Alkaline hydrolysis followed by bisulfite treatment specifically converts unmethylated cytosine to uracil, and was reported by Olek A. et al. , 1996, Nucleic Acids Res. 24:5064-6 or Frommer et al. , 1992, Proc. Natl. Acad. Sci. 5-methylcytosine remains unmodified as described by USA 89:1827-1831. The bisulfite treated DNA can subsequently be analyzed by molecular techniques such as PCR amplification, sequencing, and detection including oligonucleotide hybridization (eg, using nucleic acid microarrays). In one embodiment, the indexed nuclei within each compartment are exposed to conditions for bisulfite treatment. Bisulfite treatment of nucleic acids is well known and routine to those of skill in the art. In one embodiment, bisulfite treatment converts the unmethylated cytosine residues of CpG dinucleotides to uracil residues leaving the 5-methylcytosine residues unchanged. Bisulfite treatment results in bisulfite treated nucleic acid fragments.

After generation of the bisulfite treated nucleic acid fragment, the fragment is modified to include additional nucleotides at one or both ends (Figure 1, block 18, and Figure 2, blocks v and vi). In one embodiment, the modification comprises subjecting the bisulfite treated nucleic acid fragment to linear amplification using multiple primers. Each primer contains at least two regions; a universal nucleotide sequence at the 5'end and a random nucleotide sequence at the 3'end. The universal nucleotide sequence is the same in each primer and, in one embodiment, comprises a second sequencing primer sequence (also referred to as the read 2 primer in Figure 2) (block vii). Regions of random nucleotide sequences are used such that there should be at least one primer that is complementary to all sequences in the bisulfite treated nucleic acid fragment. The number of random nucleotides that can be used to increase the probability of full coverage to the desired level can be determined using routine methods, and can range from 6 to 12 random nucleotides, eg, 9 random nucleotides. It can be a nucleotide. In one embodiment, the number of cycles is limited to 10 cycles or less, such as 9 cycles, 8 cycles, 7 cycles, 6 cycles, 5 cycles, 4 cycles, 3 cycles, 2 cycles, or 1 cycle. The result of linear amplification is the amplified fragment-adaptor molecule. Examples of fragment-adaptor molecules are shown in FIG. Fragment-adapter molecule 30 comprises nucleotides from the transferred strand of transposome complexes 31 and 32, including transposase index and universal sequences that can be used for amplification and/or sequencing. The fragment-adapter molecule also comprises nucleotides from the nuclear 33 genomic DNA, a region of random nucleotide sequence 34, and universal nucleotide sequence 35.

After linear amplification, an exponential amplification reaction, such as PCR, is followed to further modify the ends of the fragment-adaptor molecule before immobilization and sequencing. This step results in the fragment-adapter molecule indexing by PCR (FIG. 1, block 19). The universal sequences 31, 32 and/or 35 present at the ends of the fragment-adapter molecule serve as primers and can be used for the attachment of universal anchor sequences which can be extended in amplification reactions. Two different primers are typically used. One primer hybridizes to the universal sequence at the 3'end of one strand of the fragment-adapter molecule and the second primer hybridizes to the universal sequence at the 3'end of the other strand of the fragment-adaptor molecule. .. Therefore, the anchor sequence of each primer may be different. Suitable primers can each include an additional universal sequence, such as a universal capture sequence, and another index sequence. Since each primer can contain an index, this step results in the addition of one or two index sequences, eg a second and optionally a third index. Fragment-adapter molecules with a second and optionally a third index are called dual-index fragment-adapter molecules. The second and third indexes can be inverse complements of each other, or the second and third indexes can have sequences that are not inverse complements of each other. This second index array and optionally the third index is unique to each compartment in which the distributed indexed nuclei were placed prior to treatment with sodium bisulfite. The result of this PCR amplification is a fragment or adapter molecule of multiple or library having a similar or identical composition to the fragment-adaptor molecule shown in Figure 2, block vii.

In another embodiment, the modification comprises subjecting the bisulfite treated nucleic acid fragment to conditions that result in the ligation of additional sequences to both ends of the fragment. In one embodiment, blunt end ligation can be used. In another embodiment, the fragment is, for example, Taq polymerase or Klenow exo having non-template-dependent terminal transferase activity that adds a single deoxynucleotide, such as deoxyadenosine (A), to the 3'end of the bisulfite treated nucleic acid fragment. It is prepared with a single overhanging nucleotide, depending on the activity of a particular type of DNA polymerase, such as a minus polymerase. Such enzymes can be used to add a single nucleotide "A" to the blunt-ended 3'end of each strand of the fragment. Thus, "A" can be added to the 3'end of each strand of the double stranded target fragment by reaction with Taq or Klenow exo-minase polymerase, while additional sequences added to each end of the fragment are added to the 2'end. It may include compatible "T" overhangs present on the 3'end of each region of the double-stranded nucleic acid. This end modification also prevents self-ligation of the nucleic acids, thus biasing the formation of bisulfite treated nucleic acid fragments flanking the sequence added in this embodiment.

Fragmentation of nucleic acid molecules by the methods described herein results in fragments with blunt ends and a heterogeneous mixture of 3'and 5'overhanging ends. Thus, fragment ends are repaired using methods or kits known in the art (eg, Lucigen DNA terminator end repair kit), eg, to generate optimal ends for insertion into the blunt site of a cloning vector. Is desirable. In certain embodiments, the fragment ends of the nucleic acid population are blunt ended. More specifically, the fragment ends are blunt-ended and phosphorylated. The phosphate moiety can be introduced via enzymatic treatment, eg, using a polynucleotide kinase.

In one embodiment, the bisulfite-treated nucleic acid fragment is initially labeled with identical universal adapters (also referred to as "mismatch adapters") at the 5'and 3'ends of the bisulfite-treated nucleic acid fragment. Are described in Gormley et al., US Pat. No. 7,741,463, and Bignell et al., US Pat. No. 8,053,192). It is processed by forming an adapter molecule. In one embodiment, the universal adapter contains all the sequences required for sequencing and comprises immobilizing the fragment-adapter molecule on the array. Since the nucleic acid to be sequenced is from a single cell, further amplification of the fragment-adapter molecule is useful to achieve a sufficient number of fragment-adapter molecules for sequencing.

In another embodiment, if the universal adapter does not contain all the sequences required for sequencing, a PCR step is used to further supplement the universal adapter present in each fragment-adapter molecule prior to immobilization and sequencing. It can be modified. For example, the initial primer extension reaction is performed using a universal anchor sequence complementary to the universal sequence present in the fragment-adapter molecule, where complementary to both strands of each individual fragment-adaptor molecule. An extension product is formed. Typically, PCR adds additional universal sequences, such as universal capture sequences, and another index sequence. Since each primer can contain an index, this step results in the addition of one or two index sequences, eg a second and optionally a third index, and a fragment-adapter molecule indexing by adapter ligation ( FIG. 1, block 19). The resulting fragment-adapter molecule is called dual-index fragment-adapter molecule.

A universal adapter was added by a single step method of ligating a universal adapter containing all the sequences required for sequencing, or by a two step method of ligating the universal adapter and then PCR amplifying to further modify the universal adapter. Afterwards, the final fragment-adapter molecule will contain a universal capture sequence, a second index sequence, and optionally a third index sequence. These indexes are similar to the second and third indexes described in the production of dual-index fragment-adapter by linear amplification. The second and third indexes can be anti-complementary to each other, or the second and third indexes can have sequences that are not anti-complementary to each other. These second and optional third index sequences are unique to each compartment in which the distributed indexed nuclei were placed prior to treatment with sodium bisulfite. As a result of adding universal adapters to both ends, a plurality of fragments-adaptor molecules or a library having a structure similar to or the same as the fragments-adaptor molecule 40 shown in FIG. 4 is obtained. Fragment-adapter molecule 40 comprises capture sequences 41 and 48, also referred to as 3'flow cell adapters (e.g. P5) and 5'flow cell adapters (e.g. P7'), respectively, and indices 42 and 47, such as i5 and i7. Fragment-adapter molecule 40 also includes nucleotides from the transport strand of transposome complex 43, including transposase index 44 and universal sequence 45 that can be used for amplification and/or sequencing. The fragment-adapter molecule also contains nucleotides derived from the nuclear 46 genomic DNA.

The resulting dual-index fragment-adapter molecules collectively provide a library of nucleic acids that can be immobilized and then sequenced. The term library refers to a collection of single cell derived fragments containing known universal sequences at their 3'and 5'ends.

After the bisulfite treated nucleic acid fragment has been modified to contain additional nucleotides, dual-index fragment-adapter molecules are selected for a given size range, such as 150-400 nucleotides in length, eg 150-300 nucleotides. Subject to conditions. The resulting dual-index fragment-adapter molecules can be pooled and optionally subjected to a cleaning process to increase the purity of the DNA molecule by removing at least a portion of the unincorporated universal adapter or primer. it can. Any suitable cleaning process can be used such as electrophoresis, size exclusion chromatography and the like. In some embodiments, solid phase reversibly immobilized paramagnetic beads can be used to separate desired DNA molecules from unattached universal adapters or primers and to select nucleic acids based on size. Solid-phase reversibly immobilized paramagnetic beads are available from Beckman Coulter (Agencourt AMPure XP), Thermofisher (MagJet), Omega Biotek (Mag-Bind), Promega Beads (Promega), and KapaBay (Promega), and KapaBay (Promega) and KapaBay (Promega). There is.

Multiple fragment-adapter molecules can be prepared for sequencing. After pooling the fragment-adapter molecules, they are immobilized and amplified before sequencing (Figure 1, block 20). Methods of attaching fragment-adapter molecules to substrates from one or more sources are known in the art. Similarly, methods for amplifying immobilized fragment-adaptor molecules include, but are not limited to, bridge amplification and kinetic exclusion. Methods for immobilization and amplification prior to sequencing are described, for example, by Bignell et al. (US Pat. No. 8,053,192), Gunderson et al. (International Publication No. 2016/130704 pamphlet), Shen et al. (U.S. Pat. No. 8,895,249), and Pipenburg et al. (US Pat. No. 9,309,502).

Pooled samples can be immobilized in preparation for sequencing. Sequencing can be performed as an array of single molecules or can be amplified prior to sequencing. Amplification can be performed using one or more immobilized primers. Immobilized primers can be on a flat surface or on a pool of beads. The bead pool can be separated into emulsions with a single bead in each "compartment" of the emulsion. At a concentration of only one template per "compartment", only a single template is amplified on each bead.

The term "solid phase amplification" as used herein refers to on a solid support or to a solid support such that all or part of the amplification products are immobilized on the solid support as they are formed. Refers to any nucleic acid amplification reaction performed in association with a support. In particular, this term includes solid phase polymerase chain reaction (solid phase PCR) and solid phase isothermal amplification, except that one or both of the forward and reverse amplification primers are immobilized on a solid support. Thus, the reaction is similar to standard solution phase amplification. Solid phase PCR involves the formation of an emulsion in which one primer is immobilized on beads and the other is in free solution, and colony formation in a solid phase gel matrix in which one primer is immobilized on the surface and one is in free solution. Includes the system.

In some embodiments, the solid support comprises a patterned surface. "Patterned surface" refers to the placement of different regions in or on an exposed layer of a solid support. For example, one or more regions can be a feature where one or more amplification primers are present. Features can be separated by interstitial regions where amplification primers are not present. In some embodiments, the pattern can be an xy format of features in rows and columns. In some embodiments, the pattern can be a repeating array of features and/or gap regions. In some embodiments, the pattern can be a random arrangement of features and/or gap regions. Exemplary patterned surfaces that can be used in the methods and compositions described herein are US Pat. Nos. 8,778,848, 8,778,849, and No. 9,079,148, as well as U.S. Patent Application Publication No. 2014/0243324.

In some embodiments, the solid support comprises an array of wells or depressions on the surface. It can be manufactured as is commonly known in the art using a variety of techniques including, but not limited to, photolithography, stamping techniques, molding techniques, and microetching techniques. As will be appreciated by those in the art, the technique used will depend on the composition and shape of the array substrate.

Patterned surface features include patterned covalent gels (eg, poly(N-(5-azidoacetamidylpentyl)acrylamide-co-acrylamide) (PAZAM, eg, US Patent Publication No. 2013/ Wells on glass, silicon, plastic, or other suitable solid support (see, eg, 184796, WO 2016/066586, and WO 2015/002813) (eg, , Microwells or nanowells) This process creates a gel pad used for sequencing, which gel pad may be stable over sequencing runs with multiple cycles. Covalent attachment of polymers to the wells helps maintain the gel within the structured features throughout the life of the structured substrate during various uses, however, in many embodiments, the gel is covalently attached to the wells. For example, in some conditions, silane-free acrylamides (SFAs, such as US Pat. No. 8,563,477) may be used as the gel material.

In certain embodiments, the structured substrate patterns a solid support material with wells (eg, microwells or nanowells) and the patterned support with a gel material (eg, PAZAM, SFA, or azidoyzed SFA). ) Versions (chemically modified variants thereof such as azido-SFA) coated and gel coated supports are abraded, for example by chemical or mechanical polishing, thereby retaining the gel in the well but not the well. It can be made by removing or inactivating substantially all gel from the interstitial regions on the surface of the structured substrate in between. The primer nucleic acid can be attached to the gel material. The solution of fragment-adapter molecules can then be contacted with a polished substrate such that the individual fragment-adaptor molecules are seeded into individual wells through interaction with the primer attached to the gel material. However, the target nucleic acid will not occupy the interstitial region due to the absence or inactivity of the gel material. Amplification of fragment-adaptor molecules is limited to wells, as the absence or inactivity of the gel in the stromal region prevents outward migration of growing nucleic acid colonies. This process is conveniently manufacturable, scalable, and utilizes conventional micro or nano fabrication methods.

The present disclosure encompasses "solid phase" amplification methods in which only one amplification primer is immobilized (other primers are usually in free solution), but on a solid support, immobilized forward Preferably both a primer and a reverse primer are provided. In practice, the amplification process requires an excess of primers to maintain amplification, so "multiple" identical forward primers and/or "multiple" identical reverse primers immobilized on a solid support. Will exist. References to forward and reverse primers herein should be construed as including "plurality" of such primers unless the context dictates otherwise.

As will be appreciated by those in the art, any given amplification reaction will require at least one type of forward primer and at least one type of reverse primer specific for the template being amplified. However, in certain embodiments, the forward and reverse primers can include template-specific portions of the same sequence and can have exactly the same nucleotide sequence and structure (including any non-nucleotide modifications). In other words, it is possible to perform solid phase amplification using only one type of primer, and such single primer methods are included within the scope of the invention. Another embodiment may use forward and reverse primers that contain the same template-specific sequence, but differ in some other structural features. For example, one type of primer may contain non-nucleotide modifications that are not present in the other.

In all embodiments of the present disclosure, the primer for solid phase amplification is preferably immobilized by a single point covalent bond to a solid support at or near the 5'end of the primer and the template-specific portion of the primer is , Remains free to anneal to its cognate template and the 3′ hydroxyl group is free due to primer extension. Any suitable covalent means known in the art can be used for this. The attachment chemistry chosen will depend on the nature of the solid support, and any derivatization or functionalization applied to it. The primer itself can include moieties that can be non-nucleotide chemical modifications to facilitate attachment. In certain embodiments, the primer can include a sulfur-containing nucleophile such as phosphorothioate or thiophosphate at the 5'end. In the case of solid-supported polyacrylamide hydrogels, this nucleophile can bind to bromoacetamide groups present in the hydrogel. A more specific means of attaching the primer and template to the solid support is described in WO 05/0658814, as fully described by polymerized acrylamide and N-(5-bromoacetamidylpentyl)acrylamide. Via 5'phosphorothioate attachment to a hydrogel composed of (BRAPA).

Certain embodiments of the present disclosure are, for example, "functionalized" inert substrates or matrices by the application of layers or coatings of intermediate materials that include reactive groups that allow covalent attachment to biomolecules such as polynucleotides. A solid support composed of (eg, glass slides, polymer beads, etc.) may be utilized. Examples of such supports include, but are not limited to, polyacrylamide hydrogels supported on an inert substrate such as glass. In such embodiments, the biomolecule (eg, polynucleotide) may be directly covalently bound to the intermediate material (eg, hydrogel), but the intermediate material itself is non-attached to the matrix or matrix (eg, glass matrix). It may be covalently bound. The term "covalent bond to a solid support" should therefore be construed as encompassing this type of arrangement.

Pooled samples can be amplified on beads where each bead contains a forward and reverse amplification primer. In certain embodiments, a library of fragment-adapter molecules is prepared by solid phase amplification, more specifically solid phase isothermal amplification, US Patent Application Publication No. 2005/0100900, US Patent No. 7,115,400. It is used to prepare a clustered array of nucleic acid colonies similar to those described in the specification, WO 00/18957 and WO 98/44151. The terms “cluster” and “colony” are used interchangeably herein to refer to distinct sites on a solid support that are composed of multiple identical immobilized nucleic acid strands and multiple identical immobilized complementary nucleic acid strands. To be used. The term "clustered array" refers to an array formed from such clusters or colonies. In this context, the term "array" should not be understood as requiring an ordered arrangement of clusters.

The term "solid phase" or "surface" refers to a planar array in which primers are attached to a flat surface (eg, glass, silica or plastic microscope slides or similar flow cell devices); one or two primers are attached to beads. , Beads to which the beads are amplified; or an array of beads on the surface after the beads have been amplified.

Clustered arrays are either processes of thermocycling as described in WO 98/44151 or processes in which the temperature is kept constant and the cycles of extension and denaturation are carried out with the modification of reagents. It can be prepared using Such an isothermal amplification method is described in patent application number WO 02/46456 and US patent application WO 2008/0009420. This is particularly preferred because of the lower temperatures useful in isothermal processes.

Any of the amplification methodologies described herein or generally known in the art can be used with a universal or target-specific primer to amplify an immobilized DNA fragment. Will understand. Suitable methods for amplification include Polymerase Chain Reaction (PCR), Strand Displacement Amplification (SDA), Transcription Mediated Amplification (TMA) and US Pat. No. 8,003,033, which is incorporated herein by reference in its entirety. Nucleic acid sequence-based amplification (NASBA) as described in US Pat. No. 354,354, but is not limited thereto. The amplification methods described above can be used to amplify one or more nucleic acids of interest. For example, the immobilized DNA fragment can be amplified using PCR including multiplex PCR, SDA, TMA, NASBA and the like. In some embodiments, a primer specifically directed to the polynucleotide of interest is included in the amplification reaction.

Other suitable methods for amplification of polynucleotides include oligonucleotide extension and ligation, rolling circle amplification (RCA) (Lizardi et al., Nat. Genet. 19:225-232 (1998)) and oligonucleotide ligation assays. (OLA) (generally US Pat. Nos. 7,582,420, 5,185,243, 5,679,524 and 5,573,907. EP 0 320 308 B1; EP 0 336 731 B1; EP 0 439 182 B1; WO 90/01069 pamphlet; WO 89/12696 Pamphlet; as well as WO 89/09835 pamphlet) technology. It will be appreciated that these amplification methodologies can be designed to amplify immobilized DNA fragments. For example, in some embodiments, the amplification method can include a ligation probe amplification or an oligonucleotide ligation assay (OLA) reaction that includes a primer that is specifically directed to the nucleic acid of interest. In some embodiments, the amplification method can include a primer extension-ligation reaction that includes a primer that is specifically directed to the nucleic acid of interest. As a non-limiting example of primer extension and ligation primers that can be specifically designed to amplify a nucleic acid of interest, amplification is described in US Pat. Nos. 7,582,420 and 7,611,869. As exemplified by the specification, it may include primers used for the GoldenGate assay (Illumina, Inc., San Diego, CA).

Exemplary isothermal amplification methods that can be used in the methods of the present disclosure are described, for example, in Dean et al. , Proc. Natl. Acad. Sci. USA 99:5261-66 (2002), or Multiple Isolation Amplification (MDA), as exemplified by US Pat. No. 6,214,587, for example. Not limited to. Other non-PCR based methods that can be used in the present disclosure are described, for example, in Walker et al. , Molecular Methods for Virus Detection, Academic Press, Inc. 1995; U.S. Pat. Nos. 5,455,166 and 5,130,238; and Walker et al. , Nucl. Acids Res. 20:1691-96 (1992), or strand displacement amplification (SDA), or, for example, Lage et al. , Genome Res. 13:294-307 (2003). The isothermal amplification method can be used with strand-displacing Phi 29 polymerase or Bst DNA polymerase large fragments, 5'->3' exo for random primer amplification of genomic DNA. The use of these polymerases takes advantage of their high propensity and strand displacement activity. The high kinetic properties allow the polymerase to produce fragments of 10-20 kb in length. As noted above, smaller fragments can be produced under isothermal conditions using a polymerase with low propensity and strand displacement activity such as Klenow polymerase. Further description of amplification reactions, conditions and components are described in detail in the disclosure of US Pat. No. 7,670,810.

Alternative polynucleotide amplification methods useful in the present disclosure are described, for example, in Grothues et al. Nucleic Acids Res. 21(5):1321-2 (1993), Tagged PCR using a population of 2-domain primers with a constant 5'region followed by a random 3'region. A first round of amplification is performed to allow a multiplicity of initiation for heat denatured DNA based on individual hybridizations from randomly synthesized 3'regions. Due to the nature of the 3'region, the start site appears to be random throughout the genome. The unbound primer can then be removed and the primer complementary to the constant 5'region can be used for further replication.

In some embodiments, isothermal amplification can be performed using kinetic exclusion amplification (KEA), also called exclusion amplification (ExAmp). Nucleic acid libraries of the present disclosure include reacting amplification reagents to produce a plurality of amplification sites each containing a substantially clonal population of amplicons from individual target nucleic acid seeded sites. Can be made using the method. In some embodiments, the amplification reaction proceeds until a sufficient number of amplicons are produced to fill the volume of each amplification site. In this way, filling an already seeded site to volume prevents the target nucleic acid from landing and amplifying at that site, thereby producing a clonal population of amplicons at that site. In some embodiments, apparent clonality can be achieved even if the amplification site is not filled to capacity before the second target nucleic acid reaches the site. Under some conditions, amplification of the first target nucleic acid effectively produces or creates a sufficient number of copies to overwhelm the production of copies from the second target nucleic acid that is transported to the site. You can proceed to the point where it is done. For example, in an embodiment that uses a cross-linking amplification process on circular features with diameters less than 500 nm, after 14 cycles of exponential amplification for the first target nucleic acid, contamination from the second target nucleic acid at the same site is It was determined to produce an insufficient number of contaminating amplicons to adversely affect sequencing by synthetic analysis on the Illumina sequencing platform.

In some embodiments, the amplification sites in the array can, but need not, be fully clonal. Rather, for some applications, individual amplification sites can be predominantly clustered with amplicons from the first fragment-adaptor molecule and also have low levels of contaminating amplicons from the second target nucleic acid. You can An array can have one or more amplification sites with low levels of contaminating amplicons, as long as the level of contamination does not unacceptably affect subsequent use of the array. For example, if the array is used in a detection application, acceptable levels of contamination are levels that do not affect the signal-to-noise or resolution of the detection technique in an unacceptable manner. Thus, apparent clonality is generally associated with a particular use or application of arrays produced by the methods described herein. Exemplary levels of contamination that may be tolerated at individual amplification sites for a particular application include at most 0.1%, 0.5%, 1%, 5%, 10% or 25% of the contamination amplicon. But is not limited to. The array can include one or more amplification sites with these exemplary levels of contaminating amplicons. For example, 5%, 10%, 25%, 50%, 75%, or even 100% of the amplification sites in the array may have some contaminating amplicons. At least 50%, 75%, 80%, 85%, 90%, 95% or 99% or more of the sites in the array of sites or other collections may be clonal, or may be apparently clonal Is understood.

In some embodiments, kinetic exclusion can occur when the process occurs at a rate rapid enough to effectively eliminate another event or process from occurring. For example, consider the creation of a nucleic acid array in which the sites of the array are randomly seeded with fragment-adaptor molecules from solution and copies of the fragment-adaptor molecules are generated in the amplification process to fill each of the seeded sites to capacity. According to the kinetic exclusion method of the present disclosure, the seeding and amplification process can proceed simultaneously under conditions where the amplification rate exceeds the seeding rate. In this way, the relatively rapid rate at which copies are made at the site seeded with the first target nucleic acid effectively excludes the second nucleic acid from seeding the site for amplification. The kinetic exclusion amplification method can be performed as described in detail in the disclosure of US Patent Application Publication No. 2013/0338042.

Kinetic exclusion is a relatively slow rate for initiating amplification (eg, a slow rate to make a first copy of a fragment-adaptor molecule) versus a fragment-adaptor molecule (or a first of fragment-adaptor molecule). A relatively high speed for making subsequent copies of (copy) may be utilized. In the example of the previous paragraph, kinetic exclusion results in a relatively slow rate of fragment-adapter molecule seeding (eg, relatively slow diffusion or transport), versus amplification to fill the site with a copy of the fragment-adaptor seed. It occurs due to the relatively high speed. In another exemplary embodiment, kinetic exclusion is delayed in the formation of the first copy of the fragment-adapter molecule seeded at the site (eg, delayed or slow activation), as opposed to subsequent copies. Can occur due to the relatively rapid rate at which the site is filled. In this example, several different fragment-adapter molecules may be seeded at individual sites (eg, several fragment-adapter molecules may be present at each site prior to amplification). However, the first copy formation for any given fragment-adapter molecule is randomly activated such that the average rate of first copy formation is relatively slow compared to the rate at which subsequent copies are produced. Can be done. In this case, it is possible that several individual fragment-adapter molecules were seeded at individual sites, but kinetics exclusion would only allow amplification of one of these fragment-adaptor molecules. More specifically, once the first fragment-adapter molecule has been activated for amplification, the site rapidly fills its volume with that copy, whereby a copy of the second fragment-adaptor molecule is obtained. It will prevent it being made at the site.

Amplification reagents can include additional components that promote and optionally increase the rate of amplicon formation. An example is recombinase. Recombinases may facilitate amplicons formation by allowing repeated invasion/elongation. More specifically, the recombinase can facilitate entry of the fragment-adapter molecule by the polymerase and extension of the primer by the polymerase using the fragment-adaptor molecule as a template for amplicon formation. This process can be repeated as a chain reaction in which the amplicon produced from each round of invasion/extension serves as a template in the next round. This process can be performed faster than standard PCR, as no denaturation cycle (eg, via heating or chemical denaturation) is required. As such, recombinase-promoted amplification can be performed isothermally. It is generally desirable to include ATP, or other nucleotides (or, in some cases, non-hydrolyzable analogs thereof) in the recombinase-enhanced amplification reagents to facilitate amplification. Mixtures of recombinase and single chain binding (SSB) proteins are particularly useful as SSB can further facilitate amplification. Exemplary formulations for recombinase-stimulated amplification include those sold commercially by TwistDx (Cambridge, UK) as the TwistAmp kit. Useful components of the recombinase-enhanced amplification reagents and reaction conditions are described in US Pat. No. 5,223,414 and US Pat. No. 7,399,590.

Another example of a component that can be included in an amplification reagent to promote amplicon formation and, in some cases, to increase the rate of amplicon formation is helicase. Helicases can facilitate amplicon formation by allowing the chain reaction of amplicon formation. This process can be performed faster than standard PCR, as no denaturation cycle (eg, via heating or chemical denaturation) is required. As such, helicase-promoted amplification can be performed isothermally. Mixtures of helicase and single chain binding (SSB) proteins are particularly useful as SSB can further enhance amplification. Exemplary formulations for helicase-enhanced amplification include those commercially sold as BioAmp kits from Biohelix (Beverly, MA). Further, examples of useful formulations containing helicase proteins are described in US Pat. No. 7,399,590 and US Pat. No. 7,829,284, which are incorporated herein by reference in their entirety. Has been done.

Yet another example of a component that can be included in an amplification reagent to promote amplicon formation and, in some cases, increase the rate of amplicon formation is a source binding protein.

Following attachment of the fragment-adapter molecule to the surface, the sequence of the immobilized and amplified fragment-adaptor molecule is determined. Sequencing can be performed using any suitable sequencing technique, methods for determining the sequence of immobilized and amplified fragment-adapter molecules including strand resynthesis are described in the art. Are known in, for example, Bignell et al. (US Pat. No. 8,053,192), Gunderson et al. (International Publication No. 2016/130704 pamphlet), Shen et al. (U.S. Pat. No. 8,895,249), and Pipenburg et al. (US Pat. No. 9,309,502).

The methods described herein can be used in conjunction with various nucleic acid sequencing techniques. A particularly applicable technique is that in which nucleic acids are attached to fixed locations in the array such that their relative positions do not change and the array is repeatedly imaged. The embodiment in which the image is obtained in different color channels, for example, where the images are matched with different labels used to distinguish one nucleotide base type from another is particularly applicable. In some embodiments, the process for determining the nucleotide sequence of the fragment-adapter molecule can be an automated process. Preferred embodiments include synthetic sequencing ("SBS") techniques.

SBS technology generally involves the enzymatic extension of a nascent nucleic acid strand through the repeated addition of nucleotides to a template strand. In the traditional method of SBS, a single nucleotide monomer can be provided to a target nucleotide in the presence of a polymerase at each feed. However, the methods described herein may provide more than one nucleotide monomer to the target nucleic acid in the presence of a polymerase in one delivery.

In one embodiment, the nucleotide monomer comprises locked nucleic acid (LNA) or bridging nucleic acid (BNA). If the fragment-adapter molecule is produced using one or more cytosine-depleted nucleotide sequences, such as occurs when there are cytosine-depleted nucleotide sequences in the transport chain from the transposome complex, the The melting temperature of the hybridizing nucleotide monomer changes. The use of LNA or BNA in the nucleotide monomer increases the hybridization strength between the nucleotide monomer present on the immobilized fragment-adaptor molecule and the sequencing primer sequence.

SBS can use nucleotide monomers having a terminator moiety or nucleotide monomers lacking a terminator moiety. Methods utilizing nucleotide monomers lacking terminators include, for example, pyrosequencing and sequencing using γ-phosphate labeled nucleotides, as described in further detail below. In methods using nucleotide monomers lacking terminators, the number of nucleotides added in each cycle is generally variable and depends on the template sequence and the mode of nucleotide delivery. For SBS technology that uses a nucleotide monomer with a terminator moiety, the terminator can be effectively irreversible under the sequencing conditions used, as in conventional Sanger sequencing using dideoxynucleotides, or The terminator may be reversible, as is the case with the sequencing method developed by Solexa (now Illumina, Inc.).

SBS technology can use nucleotide monomers with or without labeling moieties. Thus, uptake events can be detected based on characteristics of the label (eg, fluorescence of the label); characteristics of nucleotide monomers (eg, molecular weight or charge); byproducts of nucleotide incorporation (eg, release of pyrophosphate). In embodiments where two or more different nucleotides are present in the sequencing reagent, the different nucleotides may be distinguishable from each other, or alternatively, the two or more different labels may be labeled under the detection technique used. It can be indistinguishable. For example, the different nucleotides present in the sequencing reagents can have different labels, which will have the appropriate optics, as exemplified by the sequencing method developed by Solexa (now Illumina, Inc.). Can be distinguished using.

Preferred embodiments include pyrosequencing techniques. Pyrosequencing detects the release of inorganic pyrophosphate (PPi) when specific nucleotides are incorporated into the nascent chain (Ronaghi, M., Karamohamed, S., Petersson, B., Uhlen, M. and Nyren. , P (1996) "Real-time DNA sequencing using pyrophosphate release." Analytical Biochemistry 242(1), 84-9; , 3-11; Ronaghi, M., Uhlen, M. and Nyren, P. (1998), "A sequencing method based on real-time pyrophosphate." Science 281 (5375), 363; Nos. 6,258,568 and 6,274,320). In pyrosequencing, released PPi can be detected by immediate conversion to adenosine triphosphate (ATP) by ATP sulfatase, and the level of ATP produced is detected via luciferase-producing photons. Nucleic acids to be sequenced can be attached to features in the array and the array can be imaged to capture the chemiluminescent signal generated due to the incorporation of nucleotides in the features of the array. Images can be obtained after treating the array with a particular nucleotide type (eg, A, T, C, or G). The images obtained after addition of each nucleotide type will differ as to which features in the array will be detected. These differences in the image reflect the different array content of the features on the array. However, the relative position of each feature remains unchanged in the image. The images can be stored, processed and analyzed using the methods described herein. For example, the images obtained after processing the array with each different nucleotide type are in the same manner as exemplified herein for images obtained from different detection channels for reversible terminator-based sequencing methods. It can be handled.

In another exemplary form of SBS, cycle sequencing is described, for example, in WO 04/018497 and US Pat. No. 7,057,026, which are incorporated herein by reference in their entirety. This is accomplished, for example, by the stepwise addition of reversible terminator nucleotides containing cleavable or photobleachable dye labels, as described. This approach has been commercialized by Solexa (now Illumina Inc.) and is also described in WO 91/06678 and WO 07/123,744. The availability of fluorescently labeled terminators that can reverse termination and cleave fluorescent labels facilitates efficient cyclic reversible termination (CRT) sequencing. Polymerases can also be co-engineered to efficiently incorporate and extend from these modified nucleotides.

Preferably, in the reversible terminator-based sequencing embodiment, the label does not substantially inhibit extension under SBS reaction conditions. However, the detection label may be removable, for example by cleavage or degradation. The image can be captured after incorporation of the label into the arrayed nucleic acid features. In certain embodiments, each cycle comprises co-delivery of four different nucleotide types to the array, each nucleotide type having a spectrally distinct label. Then, four images can be acquired, each using a detection channel selective for one of four different labels. Alternatively, the different nucleotide types can be added sequentially and an image of the array can be obtained during each addition step. In such an embodiment, each image will show nucleic acid features that incorporate a particular type of nucleotide. Different features may or may not be present in different images due to the different array content of each feature. However, the relative positions of the features will remain unchanged in the image. Images obtained from such a reversible terminator-SBS method can be stored, processed and analyzed as described herein. Following the image capture step, the label can be removed and the reversible terminator moiety can be removed for subsequent cycles of nucleotide addition and detection. Removing the label after it has been detected in a particular cycle and before subsequent cycles can provide the advantage of reducing background signal and crosstalk between cycles. Examples of useful labeling and removal methods are described below.

In certain embodiments, some or all of the nucleotide monomers can include reversible terminators. In such embodiments, the reversible terminator/cleavable fluorophore may comprise a fluorophore attached to the ribose moiety via a 3'ester bond (Metzker, Genome Res. 15:1767-1776 (2005)). .. Another approach separates the terminator chemistry from the cleavage of fluorescent labels (Ruparel et al., Proc Natl Acad Sci USA 102:5932-7 (2005)). Ruparel et al. Described the development of a reversible terminator using a small 3'allyl group to block extension, but could be easily deblocked by a brief treatment with a palladium catalyst. The fluorophore was attached to the base via a photocleavable linker that can be easily cleaved by exposure to long wavelength UV light for 30 seconds. Thus, either disulfide reduction or photocleavage can be used as a cleavable linker. Another approach to reversible termination is the use of spontaneous termination that occurs after placing the bulky dye on dNTPs. The presence of charged bulky dyes on dNTPs can act as effective terminators through steric and/or electrostatic hindrance. The presence of one uptake event prevents further uptake unless the dye is removed. Cleavage of the dye removes the fluorophore and effectively reverses termination. Examples of modified nucleotides are also described in US Pat. Nos. 7,427,673 and 7,057,026, which are incorporated herein by reference in their entirety.

Additional exemplary SBS systems and methods that can be used with the methods and systems described herein are disclosed in US Patent Application Publication Nos. 2007/0166705, 2006/0188901, and No. 2006/0240439, No. 2006/0281109, No. 2012/0270305, and No. 2013/0260372, US Pat. No. 7,057,026, PCT International Publication No. 05/065814, U.S. Patent Application Publication No. 2005/0100900, and PCT International Publication No. 06/064199 and International Publication No. 07/010,251.

Some embodiments may use the detection of 4 different nucleotides using less than 4 different labels. For example, SBS can be implemented utilizing the methods and systems described in incorporated materials of US 2013/0079232. As a first example, a nucleotide-type pair is detected at the same wavelength, but based on the difference in intensity for one member of the pair compared to the other, or with the signal detected for the other member of the pair. Distinctions can be made based on changes to one member of the pair that result in the appearance or disappearance of the apparent signal (eg, via chemical, photochemical, or physical modification). As a second example, three of the four different nucleotide types can be detected under certain conditions, while the fourth nucleotide type is detectable under those conditions or their Lack of label that is minimally detected under conditions (eg, minimal detection by background fluorescence). The incorporation of the first three nucleotide types into the nucleic acid can be determined based on the presence of their respective signals, and the incorporation of the fourth nucleotide type into the nucleic acid results in the absence or minimal detection of any signal. Can be determined based on As a third example, one nucleotide type may include a label that is detected in two different channels, while other nucleotide types are detected in one or less of the channels. The above three exemplary configurations are not mutually exclusive and can be used in various combinations. An exemplary embodiment combining all three examples is a first nucleotide type detected in a first channel (eg, a label detected in a first channel when excited by a first excitation wavelength). With a second nucleotide type detected in a second channel (eg, dCTP with a label detected in a second channel when excited by a second excitation wavelength), a first and a second A third nucleotide type detected in both of the two channels (eg, dTTP with at least one label detected in both channels when excited by the first and/or second excitation wavelengths), and A fluorescence-based SBS method using a fourth nucleotide type that lacks a label that is not detected or minimally detected in either channel (eg, dGTP without a label).

In addition, sequencing data can be obtained using a single channel, as described in incorporated material of US 2013/0079232. In such a so-called 1-dye sequencing approach, the first nucleotide type is labeled, but the label is removed after the first image is generated, and the second nucleotide type is labeled with the first image. Only after being labeled. The third nucleotide type retains its label in both the first and second images, and the fourth nucleotide type remains unlabeled in both images.

Some embodiments may use sequencing by ligation techniques. Such techniques use DNA ligase to incorporate oligonucleotides and identify the incorporation of such oligonucleotides. Oligonucleotides typically have different labels that correlate with the identity of particular nucleotides in the sequence to which the oligonucleotide hybridizes. As with other SBS methods, images can be obtained after treating the array of nucleic acid features with labeled sequencing reagents. Each image will show nucleic acid features that incorporate a particular type of label. Different features may or may not be present in different images due to the different array content of each feature, but the relative position of the features will remain unchanged in the image. Images obtained from the ligation-based sequencing method can be stored, processed and analyzed as described herein. Exemplary SBS systems and methods that can be used with the methods and systems described herein are US Pat. Nos. 6,969,488, 6,172,218, and No. 6,306,597.

Some embodiments are described in Nanopore Sequencing (Deamer, DW & Akeson, M. "Nanopores and nucleic acids: Prospects foruLtrapid sequencing." Trends Biotechnol. 18, 147- 151 2000; D. Branton, "Characterization of nucleic acids by nanopore analysis." Acc. Chem. Res. 35:817-825 (2002); Li, J., M. Gershow, D. Stein, E. Brand. Golovchenko, "DNA molecules and configurations in a solid-state nanopore microscope," Nat. Mater. 2:611-615 (2003)). In such an embodiment, the fragment-adapter molecule passes through the nanopore. Nanopores can be biological pore proteins such as synthetic pores or α-hemolysin. As the fragment-adaptor molecule passes through the nanopore, each base pair can be identified by measuring the variation in the electrical conductance of the pore (US Pat. No. 7,001,792; Soni, GV. & Meller, "A. Progress towerdutrafast DNA sequencing using solid-state nanopores." Clin. Chem. 53, 1996-2001 (2007), 4; -481 (2007); Cockroft, SL, Chu, J., Amorin, M. & Ghadiri, MR, "A single-molecule nanodefects depletion activity-injection. Chem. Soc. 130, 818-820 (2008), the entire disclosures of which are incorporated herein by reference). The data obtained from nanopore sequencing can be stored, processed and analyzed as described herein. In particular, the data can be treated as an image according to the exemplary processing of optical images and other images described herein.

Some embodiments may use methods that include real-time monitoring of DNA polymerase activity. Incorporation of nucleotides is described, for example, in fluorophores as described in US Pat. Nos. 7,329,492 and 7,211,414, both of which are incorporated herein by reference. Can be detected by a fluorescence resonance energy transfer (FRET) interaction between a polymerase having a γ-phosphate labeled nucleotide or nucleotide incorporation is described, for example, in US Pat. No. 7,315,019. Using zero mode waveguides as described, fluorescent nucleotide analogs and manipulations such as those described in, for example, US Pat. No. 7,405,281 and US Pat. App. Pub. No. 2008/0108082. Detected polymerase can be used. Irradiation can be limited to the zeptoliter scale volume around the surface tethered polymerase so that incorporation of fluorescently labeled nucleotides can be observed in the low background (Levene, MJ et al., “Zero- mode waveguides for single-molecule analysis at high concentrations.", Science 299, 682-686 (2003); 1026-1028 (2008); Korlach, J. et al. (2008)). The images obtained from such methods can be stored, processed and analyzed as described herein.

Some SBS embodiments include detection of protons released upon incorporation of nucleotides into extension products. For example, sequencing based on the detection of released protons may be performed using electrical detectors and related techniques commercially available from Ion Torrent (Guilford, CT, a subsidiary of Life Technologies) or US Patent Application Publication No. 2009/0026082. The sequencing methods and systems described in 2009/0127589, 2010/0137143 and 2010/0282617 can be used. The methods described herein for amplifying target nucleic acids using kinetic exclusion can be readily applied to the substrate used to detect protons. More specifically, the methods described herein can be used to produce a clonal population of amplicons used to detect protons.

The SBS method described above may advantageously be performed in a multiplex format such that a plurality of different fragment-adapter molecules are manipulated simultaneously. In particular embodiments, different fragment-adapter molecules can be treated in a common reaction vessel or on the surface of a particular substrate. This allows for convenient delivery of sequencing reagents, removal of unreacted reagents, and detection of uptake events in multiplex fashion. In embodiments that use surface-bound target nucleic acids, the fragment-adapter molecules can be in array format. In array format, the fragment-adapter molecules can typically be bound to the surface in a spatially distinct manner. Fragment-adapter molecules can be attached by direct covalent attachment, attachment to beads or other particles, or attachment to surface-attached polymerases or other molecules. The array can include a single copy of the fragment-adapter molecule at each site (also called a feature), or multiple copies with the same sequence can be present at each site or feature. Multiple copies can be produced by an amplification method, such as cross-linking amplification or emulsion PCR, as described in further detail below.

The methods described herein are, for example, at least about 10 features/cm ² , 100 features/cm ² , 500 features/cm ² , 1,000 features/cm ² , 5,000 features/cm ² , 10,. Any of a variety of densities including 000 features/cm ² , 50,000 features/cm ² , 100,000 features/cm ² , 1,000,000 features/cm ² , 5,000,000 features/cm ² or more. An array with features at can be used.

An advantage of the method described herein is that it provides rapid and efficient parallel detection of multiple cm ² . Accordingly, the present disclosure provides an integrated system capable of preparing and detecting nucleic acids using techniques known in the art such as those exemplified above. Thus, the integrated system of the present disclosure can include fluidic components capable of delivering amplification reagents and/or sequencing reagents to one or more immobilized DNA fragments, which systems include pumps, valves, reservoirs. , Fluid lines and other components. The flow cell can be configured and/or used in an integrated system for the detection of target nucleic acids. Exemplary flow cells are described, for example, in U.S. Patent Application Publication No. 2010/0111768 and U.S. Patent Application No. 13/273,666. As illustrated for the flow cell, one or more of the fluid components of the integrated system can be used for amplification and detection methods. Taking the nucleic acid sequencing embodiment as an example, one or more fluidic components of the integrated system may be sequenced for the amplification methods described herein and in the sequencing methods as exemplified above. It can be used for delivery of determination reagents. Alternatively, the integrated system can include a separate fluid system for performing the amplification method and the detection method. Examples of integrated sequencing systems capable of producing amplified nucleic acids and sequencing the nucleic acids include MiSeq™ platform (Illumina, Inc., San Diego, Calif.) and herein by reference. It includes, but is not limited to, the devices described in incorporated US patent application Ser. No. 13/273,666.

Various compositions may occur during the performance of the methods described herein. For example, a dual-index fragment-adapter molecule comprising a dual-index fragment-adaptor molecule and a composition comprising a dual-index fragment-adapter molecule having the configuration shown in Figure 2 block vii or Figure 4 may result. A sequencing library of dual-index fragment-adaptor molecules comprising a dual-index fragment-adaptor molecule having the configuration shown in FIG. 2 block vii or FIG. 4 and a composition comprising a sequencing library can result. Such a sequencing library can be bound to an array.

The invention is illustrated by the following examples. It should be understood that the particular examples, materials, amounts, and procedures should be construed broadly in accordance with the scope and spirit of the inventions described herein.

Reagents used in Examples-Phosphate buffered saline (PBS, Thermo Fisher, Cat. 10010023)
-0.25% trypsin (Thermo Fisher, Cat. 15050057)
・Tris (Fisher, Cat. T1503)
-HCl (Fisher, Cat. A144)
-NaCl (Fisher, Cat. M-11624)
-MgCl2 (Sigma, Cat. M8226)
-Igepal (registered trademark) CA-630 (Sigma, I8896)
-Protease inhibitor (Roche, Cat. 11873580001)
PCR-clean ddH2O
-Lithium 3,5-diiodosalicylic acid (Sigma, Cat.D3635)-LAND method only-Formaldehyde (Sigma, Cat.F8775)-xSDS method-Glycine (Sigma, Cat.G8898)-xSDS method-NE buffer solution 2.1 (NEB, Cat.B7202)-xSDS method only-SDS (Sigma, Cat.L3771)-xSDS method only-Triton(TM) X-100 (Sigma, Cat.9002-93-1)-xSDS method only・DAPI (Thermo Fisher, Cat. D1306)
TD buffer from Nextera® kit (Illumina, Cat. FC-121-1031)
96 indexed cytosine-depleted transposomes (assembled using published methods, sequences shown in Table 1)
• 9-nucleotide random primer (Table 2)
・10 mM dNTP mixture (NEB, Cat. N0447)
-Klenow (3'->5' exo-) polymerase (Enzymatics, Cat. P7010-LC-L)
・200 proof ethanol ・Index added i5 and i7 PCR primers (Table 3)
-Kapa HiFi (trademark) HotStart ReadyMix
SYBR (registered trademark) Green (FMC BioProducts, Cat. 50513)
-QIAquick (registered trademark) PCR purification kit (Qiagen, Cat. 28104)
-DsDNA high sensitivity Qubit (registered trademark) (Thermo Fisher, Cat. Q32851)
・High-sensitivity bioanalyzer kit (Agilent, Cat.5067-4626)
-NextSeq sequencing kit (150-cycle high or medium)
-Unmethylated lambda DNA (Promega, Cat. D1521)
-HiSeq (registered trademark) 2500 sequencing kit (Illumina)
-HiSeq (registered trademark) X sequencing kit (Illumina)
EZ-96 DNA methylation MagPrep kit (Zymo Research, Cat D5040)
-Custom LNA sequencing primers (Table 4)
・Polyethylene glycol (PEG)
・SPRI beads

Device used in Examples 35 μM cell strainer (BD Biosciences, Cat. 352235)
• 96-well plate compatible magnetic rack • Sony SH800 cell sorter (Sony Biotechnology, Cat. SH800) or other FACS instrument capable of DAPI-based single nuclear sorting. • CFX Connect RT thermal cycler (Bio-Rad, Cat. 1855200) or other real-time thermocycler, thermomixer, Qubit® 2.0 fluorometer (Thermo Fisher, Cat. Q32866).
2100 bioanalyzer (Agilent, Cat. G2939A)
-NextSeq (registered trademark) 500 (Illumina, Cat. SY-415-1001-1)
・HiSeq (registered trademark) 2500 (Illumina)
・HiSeq (registered trademark) X (Illumina)

Oligonucleotides used in the examples

Example 1
Unmethylated lambda DNA Preparation 100 nanograms unmethylated control lambda DNA, 2X TD buffer 5 uL, NIB buffer 5uL (10mM Tris-HCl pH7.4,10MM NaCl , 3mM MgCl 2, 0.1% Igepal (Registered trademark), 1× protease inhibitor), and 4 μL of 500 nM uniquely indexed cytosine-depleted transposome were combined. The mixture was incubated at 55° C. for 20 minutes, then purified using a QIAquick® PCR purification column and eluted in 30 μL EB.

The concentration of DNA was quantified using a dsDNA sensitive Qubit 2.0 fluorometer using 2 uL of the mixture. This concentration was diluted to 17.95 pg/uL. This simulates the genomic mass of approximately 5 human cells.

Example 2
Preparation of 18% PEG SPRI Bead Mix Sera-Mag beads (1 ml) were aliquoted into low binding 1.5 mL tubes and then placed on a magnetic stand until the supernatant was clear. The beads were washed with 500 uL of a 10 mM Tris-HCl (pH 8.0) solution, the solution was removed after the supernatant became clear, and this washing step was repeated 4 times in total. The beads were resuspended in the following mixture: 18% PEG8000 (mass ratio), 1M NaCl, 10 mM Tris-HCl, pH 8.0, 1 mM EDTA, 0.05% Tween-20; at least room temperature with gentle agitation. Incubate for 1 hour, then store 18% PEG SPRI beads at 4°C. The beads were used after reaching room temperature.

Example 3
Preparation of nuclei with lithium 3,5-diiodosalicylic acid (LAND) or SDS (xSDS) NAD Preparation of Nucleation and Nucleosome Depletion When cells are in suspension cell culture, the culture is gently disrupted to disrupt cell clumps and the cells are spun at 500 xg for 5 minutes at 4°C. Pelletized and washed with 500 μL ice cold PBS.

If the cells were in adherent cell cultures, the medium was aspirated, the cells were washed with 10 ml PBS at 37°C and then at 37°C sufficient 0.25% trypsin was added to coat the monolayer. After incubation at 37° C. for 5 minutes or until 90% of the cells no longer attach to the surface, 37° C. medium was added at a 1:1 ratio to quench trypsin. Cells were pelleted by spinning at 500 xg for 5 minutes at 4°C, then washed with 500 μL ice-cold PBS.

Cells from either suspension or adherent cell cultures are pelleted by spinning at 500 xg for 5 minutes, then NIB buffer (2.5 μL 1M LIS + 197.5 μL NIB buffer). Resuspended in 200 μL of 12.5 mM LIS. After incubating on ice for 5 minutes, 800 μL of NIB buffer was added. The cells were gently passed through a 35 μM cell strainer and 5 μL of DAPI (5 mg/mL) was added.

B. Nuclear Preparation and Nucleosome Depletion xSDS Method When cells were in suspension cell culture, medium was gently triturated to disrupt cell clumps. To 10 ml cells in medium was added 406 μl 37% formaldehyde and incubated for 10 minutes at room temperature with gentle shaking. 800 microliters of 2.5 M glycine was added to the cells, incubated on ice for 5 minutes, then centrifuged at 550 xg for 8 minutes at 4°C. After washing with 10 mL ice-cold PBS, cells were resuspended in 5 mL ice-cold NIB (10 mM TrisHCl pH 7.4, 10 mM NaCl, 3 mM MgCl ₂ , 0.1% Igepal®, 1× protease inhibitor). Incubated on ice for 20 minutes with gentle mixing.

If the cells were in adherent cell cultures, the medium was aspirated, the cells were washed with 10 ml PBS at 37°C and then at 37°C sufficient 0.25% trypsin was added to coat the monolayer. After incubation at 37° C. for 5 minutes or until 90% of the cells no longer attach to the surface, 37° C. medium was added at a 1:1 ratio to quench trypsin and bring the volume to 10 ml with medium. Cells were resuspended in 10 ml medium, 406 μl 37% formaldehyde was added and incubated for 10 minutes at room temperature with gentle shaking. 800 microliters of 2.5 M glycine was added to the cells and incubated on ice for 5 minutes. Cells were centrifuged at 550 xg for 8 minutes at 4°C and washed with 10 ml ice cold PBS. After resuspending the cells in 5 mL of ice cold NIB, they were incubated on ice for 20 minutes with gentle mixing.

Cells or nuclei from either suspension cell cultures or adherent cell cultures were pelleted by spinning at 500xg for 5 minutes and washed with 900 μL of 1x NE buffer 2.1. After spinning at 500 xg for 5 minutes, the pellet was resuspended in 800 μL of 1x NE buffer 2.1 with 12 μL of 20% SDS and incubated at 42°C for 30 minutes with vigorous shaking, then 200 μL of 10% Triton ( ™ X-100 was added and incubated at 42°C for 30 minutes with vigorous shaking. The cells were gently passed through a 35 μM cell strainer and 5 μL of DAPI (5 mg/mL) was added.

Example 4
Nuclear Selection and Tagmentation Tagmentation plates were prepared with 10 μL of 1×TD buffer (for one plate: 500 μL NIB buffer+500 μL TD buffer) and 2500 single nuclei were tagged. Each well of the rotation plate. In this step, the number of nuclei per well can be varied slightly as long as the number of nuclei per well is consistent across the plate. Because the transposase index is preserved, different samples can also be multiplexed into different wells of the plate. Cells were gated according to Figure 2. After spinning down the plate at 500 xg for 5 minutes, 4OuL of 500nM uniquely indexed cytosine-depleted transposome was added to each well. After sealing, the plates were incubated for 15 minutes at 55°C with gentle shaking. The plate was then placed on ice. All wells were pooled and then passed through a 35 μM cell strainer. 5 microliters of DAPI (5 mg/mL) was added.

Example 5
Second selection of nuclei A master mix was prepared for each well with 5 uL Zymo digestion reagent (2.5 uL M-digestion buffer, 2.25 uL H2O, and 0.25 uL proteinase K). Either 10 or 22 single nuclei were sorted into each well using the most stringent sorting settings. Ten single nuclei were sorted into the wells used for the unmethylated control spike-in and 22 cells were sorted into the other wells. The plate is then centrifuged at 600 xg for 5 minutes at 4°C.

Example 6
Digestion and bisulfite conversion About 35 pg (2 uL) of unmethylated control lambda DNA pretreated with C-depleted transposomes was used to spike wells with 10 single nuclei. The plate was incubated at 50° C. for 20 minutes to digest the nuclei and 32.5 uL of freshly prepared Zymo CT conversion reagent was added according to the manufacturer's protocol. Wells were mixed by trituration and plates were centrifuged at 600 xg for 2 minutes at 4°C. The plate was placed on a thermocycler for the following steps followed by: holding at 98°C for 8 minutes, 64°C for 3.5 hours, then 4°C for less than 20 hours. Zymo MagBinding beads (5 uL) were added to each well and 150 uL of M-binding buffer was added to each well. After mixing the wells by trituration, the plate was incubated for 5 minutes at room temperature. The plate was placed on a 96-well compatible magnetic rack until the supernatant was clear.

The supernatant is removed and the wells are i) removed from the magnetic rack, ii) 100 uL of 80% ethanol is added to each well and run on the bead pellet, iii) the plate is placed back on the magnetic rack, then top It was washed with fresh 80% ethanol (by volume) by removing the clear when clear.

Desulphonation was carried out by adding 50 uL of M-desulphonation buffer to each well, thoroughly resuspending the beads by trituration, incubating for 15 minutes at room temperature, placing the plate on a magnetic rack, then the supernatant. Was achieved by clearing and removing.

The supernatant was removed. Wells i) Remove plate from magnetic rack, ii) Add 100 uL 80% ethanol to each well, run on bead pellet, iii) Put plate back on magnetic rack, then remove supernatant when clear By washing with 80% fresh ethanol (by volume).

The bead pellet was dried for about 10 minutes until the pellet began to crack visually.

Elution was achieved by adding 25 uL Zymo M-elution buffer to each well, crushing the pellet for complete dissociation, and heating the plate at 55° C. for 4 minutes.

Example 7
Linear amplification Complete eluate was used per well with the following reaction mixture: 16 uL PCR-clean H2O, 5 uL 10X NE buffer 2.1, 2 uL 10 mM dNTP mix, and 2 uL 10 uM 9-nucleotide random primers. Transferred to prepared plate.

Linear amplification was performed as follows: i) DNA was made single-stranded by incubation at 95° C. for 45 seconds, then quenched on ice and kept on ice, ii) once completely cooled, 10 U Klenow (3 '->5' exo-) polymerase is added to each well and iii) the plate is incubated at 4°C for 5 minutes, then ramped to 37°C at a rate of +1°C/15 seconds and then held at 37°C for 90 minutes. To do.

Steps i to iii were repeated 3 more times for a total of 4 rounds of linear amplification. For each amplification, the following mixture was added to the reactions in each well: 1 uL 10 uM 9-nucleotide random primer, 1 uL 10 mM dNTP mix, and 1.25 uL 4X NE buffer 2. 1. 4 rounds. Linear amplification typically significantly increases read alignment rates and library complexity compared to fewer rounds.

Wells were cleaned using the prepared 18% PEG SPRI bead mixture at 1.1X (volume concentration compared to well reaction volume) as follows. The plate was incubated for 5 minutes at room temperature, placed on a magnetic rack and the supernatant removed when clear. The bead pellet was washed with 50 uL of 80% ethanol. The remaining liquid was removed and the bead pellet was dried until cracking started. DNA was eluted in 21 uL of 10 mM Tris-Cl (pH 8.5).

Example 8
Indexed PCR Reaction Complete eluate was added to the following reaction mixture per well: 2 uL of 10 uM i7 index PCR primer, 2 uL of 10 uM i5 index PCR primer, 25 uL of 2X KAPA HiFi™ HotStart ReadyMix, and 0.5 uL of 100X SYBR. Transferred to plates prepared using ® Green I. PCR amplification was performed in a real-time thermocycler with the following cycles: 95° C. for 2 minutes (94° C. for 80 seconds, 65° C. for 30 seconds, 72° C. for 30 seconds), most of the wells were measured for SYBR. The reaction was stopped when it showed an inflection of (registered trademark) Green fluorescence. For library preparation, an inflection plateau was observed between 16 and 21 PCR cycles.

Example 9
Library Cleaning and Quantification The library was washed per well using an 18% PEGS PRI bead mixture at 0.8X (volume concentration compared to well reaction volume) as follows. The plate was incubated for 5 minutes at room temperature, placed on a magnetic rack and the supernatant was removed after clearing. The bead pellet was washed with 50 uL of 80% ethanol. The remaining liquid was removed and the bead pellet was dried until cracking started. DNA was eluted in 25 uL of 10 mM Tris-Cl (pH 8.5).

The library was pooled using 5 uL of each well and 2 uL was used to quantitate the concentration of DNA using a dsDNA Sensitive Qubit® 2.0 Fluorometer according to the manufacturer's protocol. .. The Qubit® read-out was used to dilute the library to approximately 4 ng/uL and 1 uL was run on the Sensitive Bioanalyzer 2100 according to the manufacturer's protocol. The library was then quantified for the range 200 bp to 1 kbp and the pool diluted to 1 nM for Illumina sequencing.

Example 10
Sequencing The NextSeq® 500 was set up for operation according to the manufacturer's instructions for 1 nM samples with the following modifications. The library pool was loaded at a concentration of 0.9 pM and a total volume of 1.5 mL and placed in cartridge position 10; 9 μL of 100 μM stock sequencing primer 1 was diluted in cartridge position 7 in a total of 1.5 mL of HT1 buffer. Custom primers by setting 18 μL of each custom index sequencing primer at 100 μM stock concentration into cartridge position 9 in a total of 3 mL HT1 buffer; operating NextSeq® 500 in stand-alone mode; SCIseq custom Chemistry recipe (Amini et al., 2014, Nat. Genet. 46, 1343-1349); double index selected; enter appropriate number of read cycles (150 recommended); 10 for index 1 Entered 20 cycles for Cycles and Index 2; selected custom checkboxes for all reads and indexes.

All patents, patent applications, and publications cited herein, and electronically available materials (eg, nucleotide sequence submissions in GenBank and RefSeq, and amino acid sequence submissions in SwissProt, PIR, PRF, PDB, for example). , And the translation from the annotated coding regions in GenBank and RefSeq) are incorporated herein by reference in their entirety. Supplementary materials referred to in the publication, such as supplementary tables, supplementary figures, supplementary materials and methods, and/or supplementary experimental data, are also incorporated by reference in their entirety. In the event of any inconsistency between the disclosure of the present application and the disclosure of any document incorporated herein by reference, the disclosure of the present application shall control. The foregoing detailed description and examples are provided only for clarity of understanding. Unnecessary limitations should not be understood from them. The invention is not limited to the exact details shown and described, for variations obvious to one skilled in the art are included within the invention defined by the claims.

Unless otherwise indicated, all numbers used in this specification and claims to describe the amounts of components, molecular weights, etc., are to be understood as being modified by the term "about" in all cases. is there. Therefore, unless otherwise specified, the numerical parameters described in the present specification and claims are approximate values that can be changed according to desired characteristics to be obtained by the present invention. At a minimum, and not as an attempt to limit the doctrine of equivalents to the claims, each numerical parameter is construed, at least in light of the number of significant figures reported, by applying conventional rounding techniques. Should be.

Although the numerical ranges and parameters describing the broad scope of this invention are approximations, the numerical values given in a particular example are reported as accurately as possible. However, all numerical values inherently include the ranges necessarily resulting from the standard deviation found in each test measurement.

All headings are for the convenience of the reader and should not be used to limit the meaning of the text that follows the heading, unless so specified.

Claims (86)

A method for preparing a sequencing library for determining the methylation status of nucleic acids from a plurality of single cells, said method comprising:
(A) providing an isolated nucleus from a plurality of cells;
(B) subjecting the isolated nucleus to a chemical treatment to generate a nucleosome-depleted nucleus while maintaining the integrity of the isolated nucleus;
(C) partitioning the subset of nucleosome-depleted nuclei into a first plurality of compartments containing transposome complexes, wherein each transposome complex within each compartment is a first compartment within another compartment. Distributing a first index array different from the index array of;
(D) fragmenting the nucleic acids in said subset of nucleosome depleted nuclei into a plurality of nucleic acid fragments and incorporating said first index sequence into at least one strand of said nucleic acid fragments to produce an indexed nucleus.
(E) combining the indexed nuclei to generate pooled indexed nuclei;
(F) partitioning the subset of pooled indexed nuclei into a second plurality of compartments and subjecting the indexed nuclei to bisulfite treatment to produce bisulfite treated nucleic acid fragments;
(G) The bisulfite-treated nucleic acid fragment in each compartment is amplified by linear amplification using a plurality of primers containing a general-purpose nucleotide sequence at the 5'end and a random nucleotide sequence at the 3'end to be amplified. Generating a fragment-adapter molecule;
(H) incorporating a second index sequence into the amplified fragment-adaptor molecule to produce a dual-index fragment-adapter molecule, wherein the second index sequence within each compartment is Different from the second index array within the partition, and generating;
(I) generating a sequencing library for determining the methylation status of nucleic acids from the plurality of single cells by combining the dual-index fragment-adaptor molecules;
Including the method. The method of claim 1, wherein the chemical treatment comprises treatment with a chaotropic agent capable of disrupting nucleic acid-protein interactions. The method of claim 2, wherein the chaotropic agent comprises lithium diiodosalicylate. The method of claim 1, wherein the chemical treatment comprises treatment with a detergent capable of disrupting nucleic acid-protein interactions. The method of claim 4, wherein the detergent comprises sodium dodecyl sulfate (SDS). The method of claim 5, wherein the cells are treated with a cross-linking agent prior to step (a). The method of claim 6, wherein the cross-linking agent is formaldehyde. The method according to claim 1, wherein the partitioning in steps (c) and (f) is performed by fluorescence activated nuclear sorting. 2. The method of claim 1, wherein the subset of nucleosomal depleted nuclei comprises approximately equal numbers of nuclei. 10. The method of claim 9, wherein the subset of nucleosome depleted nuclei comprises 1 to about 2000 nuclei. The method of claim 1, wherein the first plurality of compartments is a multi-well plate. The method of claim 11, wherein the multi-well plate is a 96-well plate or a 384-well plate. The method of claim 1, wherein the subset of pooled indexed nuclei comprises approximately equal numbers of nuclei. 14. The method of claim 13, wherein the subset of pooled indexed nuclei comprises 1 to about 25 nuclei. 2. The method of claim 1, wherein the subset of pooled indexed nuclei comprises at least 10 times less nuclei than the subset of nucleosomal depleted nuclei. 2. The method of claim 1, wherein the subset of pooled indexed nuclei comprises at least 100 times less nuclei than the subset of nucleosomal depleted nuclei. The method of claim 1, wherein the second plurality of compartments is a multi-well plate. 18. The method of claim 17, wherein the multi-well plate is a 96-well plate or a 384-well plate. The method of claim 1, wherein each of the transposome complexes comprises a transposase and a transposon, and each of the transposons comprises a transport chain. 20. The method of claim 19, wherein the transport chain does not contain cytosine residues. 21. The method of claim 20, wherein the transport chain comprises the first index sequence. 22. The method of claim 21, wherein the transport strand further comprises a first universal sequence and a first sequencing primer sequence. 2. The method of claim 1, wherein the bisulfite treatment converts unmethylated cytosine residues of CpG dinucleotides to uracil residues, leaving 5-methylcytosine residues unchanged. The method of claim 1, wherein the linear amplification of the bisulfite treated nucleic acid fragment comprises 1 to 10 cycles. The method of claim 1, wherein the universal nucleotide sequence at the 5'end of the primer in step (g) comprises a second sequencing primer sequence. The method of claim 1, wherein the random nucleotide sequence at the 3'end of the primer in step (g) consists of 9 random nucleotides. The incorporation of the second index sequence in step (h) contacts the amplified fragment-adapter molecule in each compartment with a first universal primer and a second universal primer, each of which comprises an index sequence. And the step of performing an exponential amplification reaction. 28. The method of claim 27, wherein the index sequence of the first universal primer is the reverse complement of the index sequence of the second universal primer. 28. The method of claim 27, wherein the index sequence of the first universal primer is different from the reverse complement of the index sequence of the second universal primer. 28. The method of claim 27, wherein the first universal primer further comprises a first capture sequence and a first anchor sequence complementary to the universal sequence at the 3'end of the amplified fragment-adaptor molecule. .. 31. The method of claim 30, wherein the first capture sequence comprises the P5 primer sequence. 28. The method of claim 27, wherein the second universal primer further comprises a second capture sequence and a second anchor sequence complementary to the universal sequence at the 5'end of the amplified fragment-adaptor molecule. .. 33. The method of claim 32, wherein the second capture sequence comprises the reverse complement of the P7 primer sequence. 28. The method of claim 27, wherein the exponential amplification reaction comprises the polymerase chain reaction (PCR). 35. The method of claim 34, wherein the PCR comprises 15-30 cycles. The method of claim 1, further comprising enriching the target nucleic acid with a plurality of capture oligonucleotides having specificity for the target nucleic acid. 37. The method of claim 36, wherein the capture oligonucleotide is immobilized on the surface of a solid substrate. 37. The method of claim 36, wherein the capture oligonucleotide comprises a first member of a universal binding pair and a second member of the binding pair is immobilized on the surface of a solid substrate. 2. The method of claim 1, further comprising selecting the dual-index fragment-adaptor molecules that fall within a given size range. 2. The method of claim 1, further comprising sequencing the dual-index fragment-adaptor molecule to determine the methylation status of nucleic acids from the plurality of single cells. A method for preparing a sequencing library for determining the methylation status of nucleic acids from a plurality of single cells, said method comprising:
(A) providing an isolated nucleus from a plurality of cells;
(B) subjecting the isolated nucleus to a chemical treatment to generate a nucleosome-depleted nucleus while maintaining the integrity of the isolated nucleus;
(C) partitioning the subset of nucleosome-depleted nuclei into a first plurality of compartments containing transposome complexes, wherein each transposome complex within each compartment is a first compartment within another compartment. Distributing a first index array different from the index array of;
(D) fragmenting the nucleic acids in said subset of nucleosome depleted nuclei into a plurality of nucleic acid fragments and incorporating said first index sequence into at least one strand of said nucleic acid fragments to produce an indexed nucleus.
(E) combining the indexed nuclei to generate pooled indexed nuclei;
(F) partitioning the subset of pooled indexed nuclei into a second plurality of compartments and subjecting the indexed nuclei to bisulfite treatment to produce bisulfite treated nucleic acid fragments;
(G) ligating the bisulfite-treated nucleic acid fragment in each compartment to a universal adapter to produce a ligated fragment-adapter molecule;
(H) incorporating a second index sequence into the ligated fragment-adapter molecule to produce a dual-index fragment-adapter molecule, wherein the second index sequence within each compartment is Different from the second index array within the partition, and generating;
(I) generating a sequencing library for determining the methylation status of nucleic acids from the plurality of single cells by combining the dual-index fragment-adaptor molecules;
Including the method. 42. The method of claim 41, wherein the chemical treatment comprises treatment with a chaotropic agent capable of disrupting nucleic acid-protein interactions. 43. The method of claim 42, wherein the chaotropic agent comprises lithium diiodosalicylate. 42. The method of claim 41, wherein the chemical treatment comprises treatment with a detergent capable of disrupting nucleic acid-protein interactions. 45. The method of claim 44, wherein the detergent comprises sodium dodecyl sulfate (SDS). 46. The method of claim 45, wherein the cells are treated with a crosslinker prior to step (a). 47. The method of claim 46, wherein the cross-linking agent is formaldehyde. 42. The method of claim 41, wherein the partitioning in steps (c) and (f) is performed by fluorescence activated nuclear sorting. 42. The method of claim 41, wherein the subset of nucleosomal depleted nuclei comprises approximately equal numbers of nuclei. 50. The method of claim 49, wherein the subset of nucleosome depleted nuclei comprises 1 to about 2000 nuclei. 42. The method of claim 41, wherein the first plurality of compartments are multiwell plates. 52. The method of claim 51, wherein the multi-well plate is a 96-well plate or a 384-well plate. 42. The method of claim 41, wherein the subset of the pooled indexed nuclei comprises approximately equal numbers of nuclei. 54. The method of claim 53, wherein the subset of pooled indexed nuclei comprises 1 to about 25 nuclei. 42. The method of claim 41, wherein the subset of pooled indexed nuclei comprises at least 10 times less nuclei than the subset of nucleosomal depleted nuclei. 42. The method of claim 41, wherein the subset of pooled indexed nuclei comprises at least 100 times less nuclei than the subset of nucleosomal depleted nuclei. 42. The method of claim 41, wherein the second plurality of compartments is a multiwell plate. 58. The method of claim 57, wherein the multi-well plate is a 96-well plate or a 384-well plate. 42. The method of claim 41, wherein each of the transposome complexes comprises a transposase and a transposon, and each of the transposons comprises a transport chain. 60. The method of claim 59, wherein the transport chain does not contain cytosine residues. 61. The method of claim 60, wherein the transport chain comprises the first index sequence. 62. The method of claim 61, wherein the transport strand further comprises a first universal sequence and a first sequencing primer sequence. 42. The method of claim 41, wherein the bisulfite treatment converts unmethylated cytosine residues of CpG dinucleotides to uracil residues, leaving 5-methylcytosine residues unchanged. 42. The method of claim 41, further comprising adding one or more nucleotides to the 3'end of the bisulfite treated nucleic acid fragment to create a 3'overhang prior to the ligation of the universal adaptor. the method of. 66. The method of claim 64, wherein the adding of one or more nucleotides is performed using terminal transferase. 65. The method of claim 64, wherein the universal adapter comprises an overhang that is reverse complementary to the 3'overhang in the bisulfite treated nucleic acid fragment. Incorporation of the second index sequence in step (h) contacts the dual-index fragment-adapter molecule in each compartment with a first universal primer and a second universal primer, each comprising an index sequence. 42. The method of claim 41, comprising performing an exponential amplification reaction. 68. The method of claim 67, wherein the index sequence of the first universal primer is the reverse complement of the index sequence of the second universal primer. 68. The method of claim 67, wherein the index sequence of the first universal primer is different from the reverse complement of the index sequence of the second universal primer. 68. The method of claim 67, wherein the first universal primer further comprises a first capture sequence and a first anchor sequence complementary to the universal sequence at the 3'end of the dual-index fragment-adapter molecule. .. 71. The method of claim 70, wherein the first capture sequence comprises the P5 primer sequence. 68. The method of claim 67, wherein the second universal primer further comprises a second capture sequence and a second anchor sequence complementary to the universal sequence at the 5'end of the dual-index fragment-adapter molecule. .. 73. The method of claim 72, wherein the second capture sequence comprises the reverse complement of the P7 primer sequence. 68. The method of claim 67, wherein the exponential amplification reaction comprises the polymerase chain reaction (PCR). 75. The method of claim 74, wherein the PCR comprises 15-30 cycles. 42. The method of claim 41, further comprising enriching the target nucleic acid with a plurality of capture oligonucleotides having specificity for the target nucleic acid. 77. The method of claim 76, wherein the capture oligonucleotide is immobilized on the surface of a solid substrate. 77. The method of claim 76, wherein the capture oligonucleotide comprises a first member of a universal binding pair and a second member of the binding pair is immobilized on the surface of a solid substrate. 42. The method of claim 41, further comprising selecting the dual-index fragment-adaptor molecules that fall within a given size range. 42. The method of claim 41, further comprising sequencing the dual-index fragment-adaptor molecule to determine the methylation status of nucleic acids from the plurality of single cells. Providing a surface comprising a plurality of amplification sites, the method comprising:
Providing the amplification site comprises at least two populations of attached single-stranded nucleic acids having free 3'ends;
Contacting the surface containing amplification sites with the sequencing library under conditions suitable to produce multiple amplification sites each containing a clonal population of amplicons from individual dual-index fragment-adapter molecules. That
41. The method of claim 40, further comprising: The number of said dual-index fragment-adapter molecules exceeds the number of amplification sites, said dual-index fragment-adaptor molecules have fluid access to said amplification sites, each of said amplification sites comprising said sequencing library 82. The method of claim 81, comprising a capacity for several dual-index fragment-adaptor molecules in. Said contacting (i) transporting said dual-index fragment-adapter molecule to said amplification site at an average transport rate, and (ii) average-amplifying said dual-index fragment-adapter molecule at said amplification site. 82. The method of claim 81, comprising simultaneously amplifying at a rate, wherein the average amplification rate exceeds the average transport rate. Providing a surface comprising a plurality of amplification sites, the method comprising:
Providing the amplification site comprises at least two populations of attached single-stranded nucleic acids having free 3'ends;
Contacting the surface containing amplification sites with the sequencing library under conditions suitable to produce multiple amplification sites each containing a clonal population of amplicons from individual dual-index fragment-adapter molecules. That
81. The method of claim 80, further comprising: The number of said dual-index fragment-adapter molecules exceeds the number of amplification sites, said dual-index fragment-adaptor molecules have fluid access to said amplification sites, each of said amplification sites comprising said sequencing library 85. The method of claim 84, comprising a capacity for several dual-index fragment-adaptor molecules in. Said contacting (i) transporting said dual-index fragment-adapter molecule to said amplification site at an average transport rate, and (ii) average-amplifying said dual-index fragment-adapter molecule at said amplification site. 85. The method of claim 84, comprising simultaneously amplifying at a rate, wherein the average amplification rate exceeds the average transport rate.